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Department of Physics, Chemistry and Biology
Bachelor’s Thesis
Sample preparation of 8-hydroxy-2’-deoxyguanosine with solid
phase extraction methodology based on molecular imprinting
polymers and conventional silica based phases
Nina Bergman
June 14, 2011
LITH-IFM-G-SE-11/2468
Link¨
opings University Department of Physics, Chemistry and Biology
581 83 Link¨
oping
Department of Physics, Chemistry and Biology
Sample preparation of 8-hydroxy-2’-deoxyguanosine with solid
phase extraction methodology based on molecular imprinting
polymers and conventional silica based phases
Nina Bergman
Thesis work done at arbets- och milj¨
omedicin, the University Hospital in
Link¨
oping
June 14, 2011
Supervisor: Per Leanderson
Examiner: Stefan Svensson
Link¨
opings University Department of Physics, Chemistry and Biology
581 83 Link¨
oping
Abstract
The aim of this study was to develop methods for sample preparation for 8-OHdG in blood
plasma samples with diﬀerent solid phase extraction techniques using HPLC with an electrochemical detector. The solid phase extraction cartridges used were Chromabond® C18,
Oasis® MAX, and three types of SupelMIP™ cartridges for chloramphenicol, riboflavin, and
nitroimidazoles. The SupelMIP™ cartridges are based on molecularly imprinted polymerstechnique. The separation of 8-OHdG in samples extracted from blood plasma was carried
out 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 for
the diﬀerent solid phase extraction methods. The highest recovery was obtained using the
Chromabond C18 cartridge with a recovery of 92%, and CV coeﬃcient 9.5% (n = 4). 8-OHdG
could not be extracted on MIP-cartridges for chloramphenicol or riboflavin, but was retained
on MIP columns for nitroimidazoles, and the highest recovery was 49%.
i
Acknowledgments
I would like to thank my supervisor Per Leanderson for all my newfound skills that I obtained
during the project, and also the rest of the employed staﬀ at Arbets- och milj¨omedicin. I would
also like to thank my examiner Stefan Svensson who always took time to help and support
me during the whole project. Thank you for all the rewarding discussions about everything.
Furthermore, I want to thank Roger S¨avenhed and Martin Josefsson at Link¨opings University
who taught me so much about analytical chemistry. Your courses provided me with enough
knowledge to work on this project.
I would also like to thank Johan Thim and Kristin Bergman for their support, Aleksandra
Kyslychenko for being a good friend, and Lorentz Larsson for helping me coming in contact with
Arbets- och milj¨
omedicin. And last, but not least, Geertruida van Maldegem for introducing
me to the world of chemistry.
ii
Abbrevations
8-OHdG 8-hydroxy-2�-deoxyguanosine
CSSA
citric acid, sodium acetate trihydrate, sodium hydroxide, acetic acid
DNA
deoxyribonucleic acid
HDV
hydrodynamic voltammogram
HPLC
high-performance liquid chromatography
MAX
mixed-mode anion-exchanger
MIP
molecularly imprinted polymers
ROS
reactive oxygen species
SPE
solid phase extraction
iii
Contents
Contents
iv
1 Introduction
1.1 8-Hydroxy-2’-Deoxyguanosine . . . . . . .
1.2 Solid Phase Extraction . . . . . . . . . . .
1.3 Molecularly Imprinted Polymers . . . . . .
1.4 High Performance Liquid Chromatography
1.4.1 Stationary and Mobile Phases . . .
1.4.2 Electrochemical Detector . . . . . .
1.5 The Aim of This Project . . . . . . . . . .
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1
1
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3
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2 Materials and Methods
2.1 Instrumentation . . . . . . . . . . . . . . . .
2.2 Chemicals . . . . . . . . . . . . . . . . . . .
2.3 High Performance Liquid Chromatography .
2.3.1 The Electrochemical Detector . . . .
2.4 Sample Preparations . . . . . . . . . . . . .
2.4.1 Aqueous Samples . . . . . . . . . . .
2.4.2 Blood Plasma Samples . . . . . . . .
2.5 Traditional Solid Phase Extraction Methods
2.5.1 Octadecyl Modified Silica Phase . . .
2.5.2 Mixed-Mode Anion-Exchanger . . . .
2.6 Commercial Molecularly Imprinted Polymers
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3 Results
3.1 HPLC Method Development . . . . . .
3.1.1 Analytical Recovery . . . . . .
3.2 Traditional Solid Phase Extraction . .
3.2.1 Octadecyl Modified Silica Phase
3.2.2 Mixed-Mode Anion-Exchanger .
3.3 Commercial MIP Cartridges . . . . . .
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4 Discussion
17
4.1 HPLC Method Development . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.2 Octadecyl Modified Silica Phase . . . . . . . . . . . . . . . . . . . . . . . 17
4.3 Mixed-Mode Anion-Exchanger . . . . . . . . . . . . . . . . . . . . . . . . 18
iv
4.4
4.5
Commercial Molecularly Imprinted Polymers . . . . . . . . . . . . . . . .
Conclusions and Future Work . . . . . . . . . . . . . . . . . . . . . . . .
18
19
Bibliography
21
Appendices
24
A Preparation of the CSSA Buﬀer
24
B Tables
25
B.1 Volume Table for SPE with C18 . . . . . . . . . . . . . . . . . . . . . . . 25
B.2 Repeated Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
v
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, amongst
other things, can also produce ROS[2]. An overproduction can lead to a state of oxidative
stress. This stress is linked to an elevated risk of oxidative damage to our DNA when the
defense system is out of balance. Oxidative damage to DNA can contribute to several
diseases such as cancer. However, our bodies try to repair the damaged DNA by enzyme
reactions[3].
One of the most reactive oxidants is the hydroxyl radical, and it can attack the nucleotides in our DNA strands, proteins, and lipids[2–4]. The hydroxyl radical can be
generated, e.g., after radiolysis of water caused by ionisation radiation, or after metaldriven dissociation of hydrogen peroxide. When the hydroxyl radical interacts with the
nucleotide 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 circulation, 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 liquid
chromatography (HPLC)[6–8], gas-chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry-mass spectrometry LC-MS/MS[6, 9], and HPLC
with enzyme-linked immunosorbent assay (ELISA)[7]. The urinary concentrations in
healthy people has been estimated to 14.7 ± 4.6 (n = 15) nmol/l[9] and the concentrations in plasma to 1–70 pg/ml[10]. In cancer patients, the concentrations are significantly
higher[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 low
concentrations for trace analysis, the sample preparation is important, and liquid-liquid
extraction (LLE) and solid phase extraction (SPE) can be helpful tools to isolate the
1
Figure 1.1: Chemical structure of 8-OHdG.
analyte from the matrix, concentrate, and simplify the chromatographic work[11]. The
SPE technique has many advantages, such as high reproducible recovery, concentration
of the analyte, requires less organic solvent than liquid-liquid extraction, and is a simple
technique 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 that
often 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 cartridge
into something called the reservoir, the frits function as filters, and the sorbent is where the sample is
adsorbed.
The analyte in the sample is either adsorbed to the sorbent material, and can be
removed selectively after the interfering compounds have been washed away, or the
analyte could pass through the sorbent while the interfering compounds are adsorbed
to 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 preparation
techniques such as SPE could be used to great advantage. For SPE analysis, C18 cartridges, 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 previous
studies. To approach the problem with low concentrations using HPLC, an electrochemical detector is most often used for detection[15].
2
SPE contains several steps: conditioning, loading of the samples, washing, and
eluting[11, pp. 556–583][12][16, pp. 2–4].
The conditioning is made to activate the sorbent and to get the maximal phase
interface between the sorbent and the sample[12]. Methanol is often used followed by
water or a buﬀer. The sample is then loaded and passed through the cartridge. For some
cartridges, the sorbent is not allowed to run dry during these steps[16, pp. 2–4]. The
flow rate is another variable that is important for some cartridges[17]. The washing step
elutes weakly bonded compounds that can interfere in the chromatographic analysis, and
the 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 a
mixed mode phase with several functionalities at the same time, which makes it possible
to obtain a greater selectivity[12]. The method development in SPE is usually based on
trial-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 sorbent, is of the same type that is often used in reversed phase HPLC columns, where
hydrophobic compounds will be adsorbed to the hydrophobic sorbent, and polar compounds will more easily pass through the cartridge. The Oasis® MAX cartridge contains
a 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 compounds, with pKa 2–8[7, 8], from plasma, aqueous, or urine samples. Since 8-OHdG is a
weak acid with its OH groups[15], and has pKa 6–8[19, 20], the Oasis® MAX cartridge
might be a good choice. According to a previous study, the Oasis® MAX cartridges
made it possible to remove interfering peaks, but with lower recovery than cartridges
containing 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 binding
sites that are imprinted in the polymer[21, 22]. This provides a lower background for
complex matrices in HPLC analysis, which makes it possible to reach lower detection
limits[22].
A template molecule, that resembles the target molecule as closely as possible, is
mixed with functional monomers. By adding a crosslinking monomer and an initiator,
a polymerization is made around the molecule. This polymerization forms a threedimensional complex around the template molecule. When removing the template
molecule, there is an imprinting of the molecule that is very specific to the target molecule
by size, shape, and to the chemical functionalities[22–24]. The process is described in
Figure 1.3.
When choosing the functional monomers it is important to know what type of interaction that is desired because the monomers decide what type of interactions there
are between the target molecule and the monomers[24]. Two commonly used functional
monomers are methacrylic acid (MAA) and 4-vinylpyridine (4-VP), both of which forms
3
Figure 1.3: Construction of a MIP. The functional monomers are mixed with the template molecule. A
crosslinker 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 also
be used to form other interactions, such as hydrogen-bonding, Van deer Waals interactions, or ionic bonding, for example[23]. Common crosslinking agents are ethylene
glycol dimethacrylate (EDMA), divinylbenzene, or N,N’-methylene bisacrylamide. A
common initiator for polymerisation is 2,2’-azobis(isobutyronitrile) (AIBN). The polymer can be packed into either ordinary SPE cartridges, or they can be connected directly
to the HPLC instrumentation as a pre-column[23], or packed in special syringes (called
MEPS), or in pipette tips[26]. Attempts to make a MIP for 8-OHdG in urine samples has
been published using HPLC with UV detection[9]. In their study, guanosine was used as
template 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 of
compounds, but no cartridge for just 8-OHdG seems to be available today. During the
research for this project, no records has been found of investigations of commercial MIP
cartridges 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 the
compounds depending on their polarity in relation to the mobile phase and stationary
phase used[11, pp. 556–583][14, pp. 313–329]. The HPLC system uses a pump system
to obtain enough pressure to make the mobile phase pass through the column at a
specific flow rate. The column contains a stationary phase of fine particles or polymers
to provide a large surface area to get high-resolution separations[11, pp. 556–583]. The
analyte is injected with a syringe into the injection valve and then introduced into the
column together with the mobile phase flow by the pressure from a pump. The analytes
are separated on a column, and will pass through the column with the mobile phase and
give diﬀerent retention times depending on the polarity for the analytes. The eluting
compounds are recognised by a detector which produce a signal that is converted by
a chromatographic software to a chromatogram with peaks whose areas corresponds to
the amount of the eluting compound.
4
1.4.1
Stationary and Mobile Phases
Reversed phase chromatography, where the mobile phase is more polar than the stationary phase, is the most commonly used technique[11, 14]. The eluent strength becomes
higher the less polar the mobile phase is[14]. The stationary phase does normally contain
silica particles bonded to carbon chains of diﬀerent lengths, usually octadecyl (C18), and
diﬀerent 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 often used in reversed phase are water or buﬀers mixed with methanol, acetonitrile, or
tetrahydrofurane (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, and
this can be a very sensitive detection principle. Moreover, the detector is sensitive to both
temperature and flow rate[11, pp. 556–583]. The sample that is eluted from the column
reaches the detector, where a potential is placed across two electrodes. Compounds that
are oxidable or reducible requires diﬀerent potentials to cause a reaction. The minimal
potential 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 for
potentials 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 in
blood plasma samples, using traditional solid phase extraction and molecular imprinting
polymers for 8-OHdG, followed by analysis with HPLC-ECD. The following questions
were 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 can
provide a higher selectivity than traditional SPE or commercial MIP cartridges?
5
Chapter 2
Materials and Methods
In this chapter, methods for sample preparation and concentration of 8-OHdG for two
traditional SPE cartridges, a C18 and a MAX cartridge, are considered. Furthermore,
three diﬀerent MIP cartridges, designed for chloramphenicol, riboflavin, or nitroimidazoles, are also tested. To investigate what had an eﬀect on the systems, the diﬀerent
steps used in SPE were varied and analysed. Aqueous samples spiked with 8-OHdG
were used in the beginning, and blood plasma samples were tested when the diﬀerent
steps seemed to work. A concentration of the sample was desired because of the low
amount found in blood plasma, so a concentration factor of ten was used in most of the
analysis.
2.1
Instrumentation
The chromatography system used was a HPLC-Jasco X-LC™ 3185 PU equipped with
Rheodyne 7125 (Cotati, California, USA) manual injector. The columns used were either
Grace Smart RP 18 3u (100 mm × 4.6 mm I.D.) or Thermo Quest Hypersil Division
ODS (250 mm × 4 mm, 3µm I.D.) with an injection volume of 10 µl. The mobile phase
was methanol:CSSA buﬀer (10:90, v/v) with a flow rate of 1.0 ml/min using Grace Smart
and 0.6 ml/min using Thermo Quest columns, respectively. The detection system used
was an ESA Coulochem II electrochemical detector. The samples were evaporated in a
Savant SpeedVac.
2.2
Chemicals
Ammonium acetate and sodium acetate trihydrate were purchased from Merck (Darmstadt, Germany); sodium hydroxide and di-sodiumhydrogenphosphate from Riedel-de
Ha¨en (Seelze, Germany); 8-hydroxy-2’-deoxyguanosine (8-OHdG), methanol, hexane,
and acetonitrile, from Sigma Aldrich (Spruce street, St.Louis, MO, USA); citric acid from
May & Baker (Dagenham, England). Blood plasma was obtained from Link¨opings University 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
6
were SupelMIP™ SPE cartridges made for chloramphenicol, 55 µm, 25 mg/10 ml (53240U), 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 mobile
phases were made with 6%, 8%, and 10% methanol/CSSA buﬀer, 2% acetonitrile/CSSA
buﬀer, 0.1% acetic acid and 6% methanol/CSSA buﬀer, and analysed under isocratic
conditions. 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 were
also tested.
2.3.1
The Electrochemical Detector
To obtain a HDV for 8-OHdG, the voltage on the detector was set to diﬀerent values
between 100–600 mV, and samples with the same concentration were injected into the
HPLC-system as the current (nA) and peak areas were registered.
2.4
2.4.1
Sample Preparations
Aqueous Samples
A stock solution of 8-OHdG with a concentration of 10 µmol/l was made in water. From
this solution, a working solution with the concentration of 100 nmol/l was prepared in
mobile phase, or buﬀer.
2.4.2
Blood Plasma Samples
To precipitate the proteins, 110 µl of cold 5% trichloroacetic acid was added to 1000 µl
of both a spiked plasma sample containing 100 nmol/l 8-OHdG and an unspiked plasma
sample. The samples were vortexed for 15 seconds and centrifuged at 14100g for 2 minutes. 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 acetate
with pH 5.25.
2.5
2.5.1
Traditional Solid Phase Extraction Methods
Octadecyl Modified Silica Phase
Several measurements with diﬀerent conditions were made to develop a sample preparation method with C18 cartridges to see which conditions were the most suitable. This
was accomplished by trying to separate the peaks and calculating the recovery for 8OHdG in aqueous and blood plasma samples. The conditions investigated were diﬀerent
SPE cartridges, washing, and eluents. According to a previous study, 8-OHdG in urine
7
samples has been successfully extracted using C18 cartridges[29]. Using their proposed
method as a starting point, several tests were carried out with diﬀerent variables; see
Table B.1 in the Appendix for specifics. Tests were made to see if acetonitrile or hexane
could work as washing solution, and if acetonitrile could be used as eluent.
The final method obtained was the following: The C18 cartridges were first preconditioned with 1000 µl methanol followed with 1000 µl 25 mmol/l phosphate buﬀer
with pH 5.5. The sample, 1000 µl for aqueous samples and approximately 700 µl for
blood plasma samples, was then loaded into the cartridge and washed with 300 µl of
the 25 mmol/l phosphate buﬀer and 300 µl water. The cartridges were dried with
nitrogen gas and placed in a SpeedVac at 40°C for 15 minutes. Elution was performed
with 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, and
centrifuged at 14100g for one minute. The evaporation step is made to concentrate the
amount of 8-OHdG by a factor ten in the samples. See Figure 2.1 for a sketch of the
proposed SPE method.
Figure 2.1: The proposed SPE method using C18 cartridges. First the cartridge is conditioned with
methanol and phosphate buﬀer. The sample is then loaded into the cartridge. The cartridge is washed
with 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 as
a third washing step to try to dispose of peaks that elute late, and to get a shorter analysis
time. Repeated extractions were made with five blood plasma samples spiked with 100
nmol/l 8-OHdG to investigate the variation between the samples. These samples were
prepared using 250 µl plasma for each sample, the remaining chemicals were rescaled to
match the amount of blood plasma.
Another extraction, using 500 µl spiked blood plasma and chemicals rescaled to
match this volume, was injected and analysed four times, to investigate the variation
within the samples.
8
2.5.2
Mixed-Mode Anion-Exchanger
In a previous study[6], an Oasis® MAX cartridge has been used successfully to remove
interfering peaks. Diﬀerent pH values of the samples and washing solutions were tested
to see if pH has an eﬀect on the loading and washing systems. Aqueous samples were
made 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. Each
cartridge was then washed with 1000 µl of the 25 mmol/l phosphate buﬀer which has
the 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 that
provides the first fraction is important, one sample was forced through the cartridge at a
high speed, and one was allowed to pass through the cartridge slowly. The first fractions
were collected and analysed. Investigations were also made to see if the concentration of
the buﬀers had any eﬀect. This was accomplished by making new samples of 100 nmol/l
8-OHdG: one in 1 mmol/l ammonium acetate buﬀer and one in 25 mmol/l phosphate
buﬀer.
The final method obtained was the following: The cartridges were conditioned with
2000 µl methanol and 2000 µl Milli-Q water. The blood plasma samples were loaded into
the cartridge and washed respectively with 1000 µl phosphate buﬀer, pH 5, and 1000 µl
methylene 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 then
resuspended 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 cartridges were tested: SupelMIP™ SPE cartridges for chloramphenicol, riboflavin, or
nitroimidazoles. 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, and
later blood plasma samples were tested on the nitroimidazole cartridge. The steps
below follows those proposed in the data sheets for the respective cartridge; see [30–32],
respectively.
SupelMIP™ SPE Chloramphenicol. The cartridges were conditioned with 1000 µl
methanol, 1000 µl milli-Q water, and the samples were loaded into the cartridges. During
these steps, the cartridges were not allowed to dry. 8-OHdG was eluted with 2 × 1000 µl
methanol:acetic acid:water (89:1:10, v/v/v) and the first fraction for each sample was
analysed on the HPLC to see which amount of 8-OHdG that was retained in the cartridge.
SupelMIP™ SPE Riboflavin. The cartridges were conditioned with 1000 µl
methanol, 1000 µl milli-Q water, and the cartridges were not allowed to dry during
these 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.
9
SupelMIP™ SPE Nitroimidazoles. The cartridges were conditioned with 1000 µl
toluene, 1000 µl acetonitrile, and 1000 µl 10 mmol/l ammonium acetate with pH 6. The
samples, 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-Q
water, 2×1000 µl hexane, and 1000 µl heptane:toluene (3:1, v/v). Between each washing
step, 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°C
over the night, and resuspended in 100 µl mobile phase consisting of methanol:CSSA
buﬀer (10:90, v/v).
Tests were made with diﬀerent conditioning solvents, similar to those used for the
nitroimidazole cartridge, on the riboflavin and chloramphenicol cartridges, and attempts
to elute 8-OHdG with methanol, or acetonitrile, with 0.5% acetic acid were made on
the nitroimidazole cartridge to see if the evaporation time in the SpeedVac could be
reduced with a diﬀerent eluent. To see if the sample loading volume was important, one
blood plasma sample was diluted with 100 µl 1 mmol/l ammonium acetate buﬀer and
one with 500 µl 1 mmol/l ammonium acetate buﬀer.
10
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 rates
were investigated. The potentials were plotted against the noted peak areas in an HDV
graph; see Figure 3.1. The information from the HDV shows that the optimal signal to
noise ratio is near 300 mV. This potential gives a complete oxidation of 8-OHdG and
was 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 the
peak 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 Grace
Smart column, and the flow rate set to 1 ml/min, resulted in a retention time of 4.6 minutes. However, when blood plasma samples were used, there was an interfering peak
in the chromatogram at almost the same retention time as 8-OHdG. Attempts to obtain a better separation of the peaks by changing the flow rate, and changing the mobile
11
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 phase
with acetonitrile:CSSA buﬀer (2:98, v/v) resulted in a retention time of 5.59 minutes
and no improvement of the separation compared with 6% methanol. When changing
the mobile phase to methanol:CSSA buﬀer (10:90, v/v), using Thermo Quest Hypersil
Division ODS, instead with a flow rate of 0.5 ml/min, the 8-OHdG peak was separated
from 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 blood
plasma samples. A Chromatogram of 8-OHdG, separated on the Thermo Quest column
using 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 peaks
are separated on the Thermo Quest Hypersil Division ODS (250 mm × 4 mm, 3µm I.D.) column, at a
flow 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 E1
to 150 mV. This had no significant eﬀect on which compounds, interfering or not, that
were detected.
3.1.1
Analytical Recovery
In the absence of an internal standard, the recovery of 8-OHdG is calculated using
the peak area ratio of the sample using the SPE method of interest, to one known
reference sample spiked with 100 nmol/l 8-OHdG in buﬀer. Since the evaporation and
resuspendation enrich the samples, the peak area of the reference samples were multiplied
by the corresponding concentration factor. The analysis of the aqueous reference samples
were carried out on the same day, and under the same conditions, as for the blood plasma
samples.
12
3.2
3.2.1
Traditional Solid Phase Extraction
Octadecyl Modified Silica Phase
The C18 method provided a high recovery, and 8-OHdG did not pass through in the
first fraction or with the first washing step of the cartridges. Washing with hexane gave
no loss in recovery in aqueous samples, but tests with blood plasma did not show any
improvement of washing away late eluting peaks in the chromatogram. Washing with
acetonitrile 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 removed a considerable amount of background in the latter part of the chromatogram; see
Figure 3.3.
Figure 3.3: Chromatogram of one spiked blood plasma sample (100 nmol/l 8-OHdG) using solid phase
extraction on a C18 SPE cartridge, and one diluted blood plasma sample (blood plasma:1 mol/l ammonium acetate buﬀer, 1:1, v/v) without SPE. The black line shows the spiked blood plasma sample, using
SPE with a C18 cartridge, and the purple line shows the blood plasma without SPE. The retention
time for 8-OHdG in the spiked blood plasma sample was 4.7 min. The HPLC column used was Grace
Smart RP 18 3u (100 mm × 4.6 mm I.D.) with a flow rate of 1.0 ml/min, and mobile phase consisting of methanol:CSSA buﬀer (8:92, v/v). The peaks are not separated under these chromatographic
conditions.
In Table 3.1, the peak areas for each step of the extraction and calculated recovery for
aqueous samples spiked with 100 nmol/l 8-OHdG is shown; see Table B.1 in Appendix for
specifics of the diﬀerent tests. In blood plasma samples, hexane was tested as a washing
step to see if the peaks that elute late could be avoided so a shorter analysis time could
be obtained, but the results showed no significant diﬀerence to chromatograms without
hexane. 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 C18
cartridge. The volume of the spiked aqueous sample used in these tests is 900µl, so the concentration
factor is nine. The table shows the peak areas of the reference sample with 100 nmol/l 8-OHdG before
extraction; in the first fraction; in wash 1–3; in the eluent after extraction, evaporation, and redilution
of the sample in 100 µl mobile phases; and the calculated recovery for each test.
Test
1a
1b
2a
2b
2c
3a
3b
3c
3d
Ref. sample
150
150
156
156
156
163
163
163
207
Frac 1 Wash 1 Wash 2 Wash 3 Elution
833
166
1274
2
2305
2000
1536
1631
1742
1648
2191
Recovery (%)
12
94
164
142
109
111
119
112
118
To examine the variation in measurements, five blood plasma samples were prepared
with concentration 100 nmol/l 8-OHdG. Assuming that the measurements follow a normal distribution, the confidence interval
64.9 < µ < 75.0
(%)
was obtained for the recovery µ using a confidence degree of 95%. See Table B.2a
in 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 times
to investigate how much the measurements varies within the same sample. Assuming a
normal distribution again, a 95% confidence interval for the recovery µ was calculated
to 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 pH
values, was that everything was trapped in the cartridges independently of pH value. In
further aqueous solutions, pH 5 was chosen. When using a syringe to force the sample
through the cartridge, 17% passes through without bonding to the cartridge, and can be
seen in the first fraction. The concentration of the buﬀer had no detectable eﬀect on the
sample loading. In one of the tests, where hexane was used in the washing step, there was
a loss of 7% 8-OHdG that passed through with the hexane. Using methylene chloride
as the washing solvent instead caused no loss, so this was used in further analysis as
the washing step. Eluting one aqueous sample with methanol and 0.5% acetic acid gave
the 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 extracted
with 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 solid
phase extraction on a MAX cartridge. The HPLC column used was Thermo Quest Hypersil Division
ODS (250 mm × 4 mm, 3µm I.D.) with a flow rate of 0.6 ml/min, and mobile phase consisting of
methanol:CSSA buﬀer (8:92, v/v). There is a ridge at the end of the chromatogram, which indicates
that 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 riboflavin
cartridge, 62% 8-OHdG was retained. and to the chloramphenicol cartridge, 50% was
retained. However, when the cartridges for nitroimidazoles were used, 100% of the 8OHdG was retained. To see if the riboflavin and chloramphenicol cartridges could work
with conditions similar to those used with the nitroamidozole cartridge, the same preconditioning procedure was used for those cartridges. On the chloramphenicol cartridge,
only 29% was retained, and on the riboflavin cartridge, 45% was retained. Since it did
not work suﬃciently well, it was decided to do further analysis only on the nitroimidazole
cartridge. Tests to elute 8-OHdG with acetonitrile:water (60:40, v/v) and 0.5% acetic
acid, methanol with 0.5% acetic acid, and acetonitrile with 0.5% acetic acid, gave the
recoveries 44%, 86%, and 47%, respectively; see Table 3.2.
The use of methanol with 0.5% acetic acid also reduced the evaporation time from
one night to one hour, so all further elutions were made with methanol and 0.5% acetic
acid since it also provided the highest recovery in the aqueous samples. After the first
washing 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-OHdG
extracted 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 sample
in 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 acetonitrile
and 0.5% acetic acid, respectively, were tested.
Test
A
B
C
Ref. sample
152
152
152
Frac 1 Wash 1 Wash 2 Wash 3 Elution
26
209
6
668
89
14
1310
196
6
708
Recovery
44%
86%
47%
the water through the cartridge in one of the samples. In the second and third washing
step, there is too much loss, so these washing steps are eliminated. In blood plasma
samples, the samples that were diluted with 100 µl and 500 µl 1 mmol/l ammonium
acetate buﬀer gave the recoveries 13% and 49%, respectively. A chromatogram using
nitroimidazole 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 solid
phase extraction with MIP nitroimidazole cartridges. The column used was Thermo Quest Hypersil
Division ODS (250 mm × 4 mm, 3µm I.D.) with at a flow rate of 0.5 ml/min, and mobile phase
consisting 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 if
suﬃcient separation of the peaks could be obtained in blood plasma samples. This was
not successful due to the complex matrix. Many of these tests are left out of the report
since they did not contribute to any important progress.
According to a previous study[10], 8-OHdG has been separated from plasma samples
using a Develosil C30 column. There was no access to any type of HPLC column with
a C30 stationary phase, so other columns were tested instead. One such column was
the Thermo Quest Hypersil Division ODS (250 mm × 4 mm, 3µm I.D.), which made it
possible to separate the peaks with an approximate retention time of 18 minutes using
a flow rate of 0.5 ml/min. The time of analysis was quite long for the blood plasma
samples, about 35 minutes per analysis. One possible solution could be to use gradient
separation, and increase the concentration of methanol later in the chromatogram. This
could decrease the time of analysis. However, this method will also require some time
for 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 variation, even within the same sample. The recoveries in the aqueous samples are a lot
higher than 100% in some cases, which could be an eﬀect from the variation. To provide
an accurate analysis in future work, an internal standard is needed. One possible reason
for the variation is the manual injection, so an auto injector should be able to provide a
lower variation. Another reason can be because of the low amount of blood plasma that
was used in the tests, but since there was a lack of blood plasma during this analysis
there was no possibility to use larger amounts for each extraction. The pipettes may
also have contributed to some quantity of the variation.
Since there was no access to a suitable internal standard during the analysis in this
project, 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, shows
that 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 were
varied, but it was found that pH had no eﬀect on the bonding of 8-OHdG in aqueous
solutions. All 8-OHdG was retained in the sorbent in the cartridge independently of pH
value. This indicates that only the reversed phase eﬀect is active. A better result could
possibly had been obtained in plasma samples if also the anion eﬀect had been activated
by 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 in
the matrix could perhaps had been broken. In the analysis, approximately pH 5 was
used. Hence, only the reversed phase eﬀect was active. A combined eﬀect, from both
reversed 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 sufficiently well to remove the nonpolar compounds. This causes the analysis to take
approximately 10 minutes longer compared to SPE made with the C18 cartridge, since
the base line takes longer to stabilise between the diﬀerent analyses. The recovery is less
than that obtained for C18, but the peaks are still separated and there are no interfering
peaks 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 the
time 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 them
actually worked for 8-OHdG. Since MIP cartridges are tailor-made to match a specific
compound, or a group of similar compounds, it is surprising that recovery of 8-OHdG in
plasma samples was high when using the nitroimidazole cartridge. The discovery that
elution with methanol and 0.5% acetic acid could be made instead of using the proposed
acetonitrile:water (60:40, v/v) solution is very useful since it reduces the evaporation
time from one night to one hour. The water does not seem to have a great eﬀect on
the 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 suﬃciently well, even for aqueous samples. The reason for this might be that the molecules
are so diﬀerent, both in size and other properties, that 8-OHdG does not bind to the
sorbent in these cartridges. Therefore, these cartridges were not used for blood plasma
samples.
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 one
suggested previously. The loading and washing step had a recommended flow rate of
less than 0.5 ml/min, or to use gravity flow if possible. In this study, gravity flow
was applied. This process was time consuming and complicated due to the fact that
the sorbent was not allowed to dry during the conditioning of cartridge. It is also not
clear if everything was eluted when using this eluent, so further analysis is necessary
to ensure that everything is extracted from the cartridge so following samples are not
contaminated by leftover substances if the cartridges are used more than once.
Another phenomenon that was somewhat surprising was the fact that blood plasma
samples that were diluted provided a higher recovery than the corresponding undiluted
ones. One possible explanation could be that in the undiluted sample, proteins and other
compounds interfere more when 8-OHdG is attached to binding sites in the cartridge.
The chromatograms for blood plasma samples using the nitroimidazole cartridge
shows that all of the interfering compounds have been removed; see Figure 3.5. Compared with the chromatogram where the MAX cartridge was used, this one is better due
to the lack of the ridge at the end, and therefore has a shorter time of analysis. But
still, 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 aqueous
samples, which was expected. One possible reason for this is that the complex matrix
probably takes binding sites from 8-OHdG in the cartridges. Another reason for the loss
of recovery in blood plasma samples is that 8-OHdG could be aﬀected by the protein
precipitation when trichloroacetic acid was used. One test was made to try to ultrafiltrate the blood plasma instead, using a membrane with a molecular weight cut oﬀ
at 10000 dalton, but the filter broke because of the centrifugal forces. A diﬀerent type
of filter is necessary, but was not available at the time. There should be more analysis
made with filtration, or alternative precipitation techniques, to see if this has an eﬀect
on the recovery.
The C18 cartridge was the most straight-forward sample preparation method to work
with. The flow rate had no significant eﬀect and the cartridge was allowed to run dry
between the steps, so the laboratory work was less demanding. The MAX cartridge
was more time consuming since the flow rate of the sample loading had an eﬀect on the
recovery. Furthermore, the method requires several chemicals that are toxic and bad
for the environment, and it was necessary to carry out the extractions under a fume
hood. The nitroimidazole MIP cartridge was the most diﬃcult to work with for several
reasons. The cartridge was not allowed to run dry between steps, and for some parts
of 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 for
8-OHdG, it should have been possible to achieve a higher selectivity for 8-OHdG and to
obtain a greater recovery.
19
Since there was variation, an internal standard, and an auto injector, is probably
needed in further analysis. The variation could also have been aﬀected by the low
volumes of blood plasma used during those tests. Investigations using a larger amount
of blood plasma may reduce the variation caused by the the pipettes. An automation of
the SPE method is another alternative to reduce the variation between the samples by
lowering the operator errors[16, pp. 243–244]. The fact that the variation within the same
sample was higher than the variation between the samples remains unexplained. All of
the 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 solutions are multiplied by the concentration factor. One should probably use a reference
solution of the same concentration as the enriched sample instead of using a multiplication 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 possibility that the method may vary between diﬀerent people. To be able to make a
quantitative analysis in patients, the limit of quantification has to be determined to
see if the proposed method with C18 could work with the chromatographic conditions
obtained. A calibration curve should be derived for spiked blood plasma samples, so
that the concentration can be estimated in unknown blood plasma samples. If it turns
out not to be possible to detect low enough concentrations using SPE with C18 in blood
plasma samples, one should look into constructing a MIP to remove more of the matrix
and 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 the
current literature is needed to see what type of crosslinking agent, functional monomers,
initiator, solvents, and template molecule, that can be used. A template molecule must
be chosen to be as similar as possible to the target molecule, which in this case is
8-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 be
picked carefully. One must consider what types of interactions that are desirable is this
case, to obtain a high selectivity for 8-OHdG in blood plasma samples. When it is decided
what chemicals to use, a suitable cartridge, pipette, pre-column, or similar object, has
to be chosen to contain the molecular imprinted polymer. After this, it is possible to
calculate the amounts of chemicals that are needed. When the MIP is completed, one
can proceed with method development, e.g., testing diﬀerent conditioning steps, washing
steps, and eluents.
20
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[29] Sabatini, L. et al, A method for routine quantitation of urinary 8-hydroxy-2deoxyguanosine based on solid-phase extraction and micro-high-performance liquid
chromatography/electrospray ionization tandem mass spectrometry, Rapid Communications in Mass Spectrometry 19 (2004), no. 2, 147–152.
[30] Supelco instruction set (data sheet), SupelMIP™ SPE - Chloramphenicol, SigmaAldrich 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.
[32] Supelco instruction set (data sheet), SupelMIP™ SPE - Nitroimidazoles, SigmaAldrich Co. 2009. http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/
Datasheet/t709075.pdf; verified 2011-05-22.
23
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 to
get the best conditions and recovery of 8-OHdG in aqueous samples for the C18 cartridge. A is 900 µl
methanol, B is 300 µl water, C is 300 µl 25 mM phosphate buﬀer with pH 5.5, D is 100 µl mobile phase
with methanol:CSSA buﬀer (8:92, v/v), E is 900 µl 25 mM phosphate buﬀer with pH 5.5, F is 300 µl
acetonitrile, G is 300 µl hexane, and H is 300 µl methanol.
Test
1a
1b
2a
2b
2c
3a
3b
3c
3d
Conditioning Wash 1 Wash 2 Drying Wash 3 Elution
A+E
C
B
Yes
F
2×H
A+E
C
B
Yes
2×H
A+E
C
B
Yes
G
2×H
A+E
C
B
Yes
2×H
A+E
C
B
Yes
2×F
A+E
C
B
Yes
G
2×H
A+E
C
B
Yes
G
2×H
A+E
C
B
Yes
2×H
A+E
C
B
Yes
2×H
25
Resuspendation
D
D
D
D
D
D
D
D
D
B.2
Repeated Tests
Table B.2: Repeated tests for the C18 cartridge with 100 nmol/l 8-OHdG in blood plasma samples.
Between samples
Peak area Recovery (%)
930
68
967
70
896
65
1045
76
971
71
Within samples
Peak area Recovery (%)
1200
87
1270
92
1090
79
1027
75
(b) Analysis of the same blood
plasma sample four times. Concentration 100 nmol/l.
(a) Analysis of five diﬀerent blood
plasma samples with the concentration 100 nmol/l.
26