novel solid phase extraction and mass spectrometry approaches …311611/fulltext01.pdf · 2010. 5....

Novel Solid Phase Extraction and Mass Spectrometry Approaches

to Multicomponent Analyses in Complex Matrices

Nahid Aminiناهېد امېنى

Doctoral ThesisDepartment of Analytical Chemistry

Stockholm University2010

Academic dissertation for the Degree of Doctor of Philosophy in Analytical Chemistry at Stockholm University to be publicly defended on Wednesday 2nd of June 2010 at 10:00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, Sweden.

© Nahid Amini, Stockholm 2010ISBN 978-91-7447-050-5Printed by Universitetsservice US-AB, Stockholm, Sweden

To Arvid and Alvin

4 Abstract

AbstractAnalysis of compounds present in complex matrices is always a challenge, which can be partly overcome by applying various sample preparation techniques prior to detection. Ideally, the extraction techniques should be as selective as possible, to minimize the concentration of interfering substances. In addition, results can be improved by efficient chromatographic separation of the sample components. The elimination of interfering substances is especially important when utilizing mass spectrometry (MS) as a detection technique since they influence the ionization yields. It is also important to optimize ionization methods in order to minimize detection limits.In the work this thesis is based upon, selective solid phase extraction (SPE) materials, a restricted access material (RAM) and graphitized carbon black (GCB) were employed for clean-up and/or pre-concentration of analytes in plasma, urine and agricultural drainage water prior to liquid chromatography/mass spectrometry (LC/MS). Two SPE formats, in which GCB was incorporated in µ-traps and disks, were developed for cleaning up small and large volume samples, respectively. In addition, techniques based on use of sub-2 µm C18 particles at elevated temperatures and a linear ion trap (LIT) mass spectrometer were developed to improve the efficiency of LC separation and sensitivity of detection of 6-formylindolo[3,2-b]carbazole (FICZ) metabolites in human urine.It was also found that GCB can serve not only as a SPE sorbent, but also as a valuable surface for surface-assisted laser desorption ionization (SALDI) of small molecules. The dual functionality of GCB was utilized in a combined screening-identification/quantification procedure for fast elimination of negative samples. This may be particularly useful when processing large numbers of samples. SALDI analyses of small molecules was further investigated and improved by employing two kinds of new surfaces: oxidized GCB nanoparticles and silicon nitride.

5List of publications

List of publicationsThis thesis is based upon the following publications, which are referred to in the text by the corresponding Roman numerals. For convenience, the studies described in Papers I-VI are referred to as Studies I-VI, respectively.

I Feasibility of an On-Line Restricted Access Material/Liquid Chromatography/Tandem Mass Spectrometry Method in the Rapid and Sensitive Determination of Organophosphorus Triesters in Human Blood PlasmaNahid Amini, Carlo CrescenziJournal of Chromatography B 2003, 795: 245-256

II μ-TrapfortheSALDI-MSScreeningofOrganicCompoundsPrior to LC/MS AnalysisMohammadreza Shariatgorji, Nahid Amini, Gunnar Thorsen, Carlo Crescenzi, Leopold L. IlagAnalytical Chemistry 2008, 80: 5515-5523

III ScreeningandQuantificationofPesticidesinWaterUsingaDual-Function Graphitized Carbon Black Disk Nahid Amini, Mohammadreza Shariatgorji, Carlo Crescenzi, Gunnar ThorsenAnalytical Chemistry 2010, 82: 290-296

IV The Suggested Physiologic Aryl Hydrocarbon Receptor Activator and Cytochrome P4501 Substrate 6-Formylindolo[3,2-b]carbazole is Present in HumansEmma Wincent, Nahid Amini, Sandra Luecke, Hansruedi Glatt, Jan Bergman, Carlo Crescenzi, Agneta Rannug, Ulf RannugJournal of Biological Chemistry 2009, 284:2690-2696

6 List of publications

V SALDI-MSSignalEnhancementUsingOxidizedGraphitizedCarbon Black NanoparticlesNahid Amini, Mohammadreza Shariatgorji, Gunnar ThorsenJournal of the American Society for Mass Spectrometry 2009, 20:1207-1213

VI Silicon Nitride Nanoparticles for Surface-Assisted Laser Desorption/Ionization of Small Molecules Mohammadreza Shariatgorji, Nahid Amini, Leopold L. IlagJournal of Nanoparticle Research 2009, 11:1509-1512

The contributions of the author of this thesis to these studies were as follows:Paper I: involved in developing the idea, responsible for all experiments and writing the major part of the paper.Papers II, III and V: involved in developing the idea, responsible for all experiments and writing the major part of the papers (all equal contributions with the first/second author).Paper IV: responsible for the LC/MS parts of the study: idea, experiments and writing.Paper VI: responsible partly for developing the idea, experiments and writing of the paper.

The author also contributed to the following papers, but they are not included in the thesis. Matrix-LessLaserDesorption/IonisationMassSpectrometry

ofPolyphenolsinRedWineZdeněk Spáčil, Mohammadreza Shariatgorji, Nahid Amini, Petr Solich, Leopold L. Ilag Rapid Communications in Mass Spectrometry 2009, 23:1834-1840

7List of publications

An Enzymatic Flow Analysis Method for the Determination of Phosphatidylcholine in Sediment Pore Waters andExtractsNahid Amini, Ian McKelvieTalanta 2005,66: 445-452

Structural Studies of Ambient Temperature Plastic Crystal Ion ConductorsD. R. MacFarlane, P. Meakin, N. Amini, M. ForsythJournal of Physics: Condensed Matter 2001,13: 8257-8267

Pyrrolidinium Imides: A New Family of Molten Salts and Conductive Plastic Crystal PhasesD. R. MacFarlane, P. Meakin, J. Sun, N. Amini, M. ForsythJournal of Physical Chemistry B 1999,103: 4164-4170

8 Abbreviations

Abbreviations1DLC One-dimensional liquid chromatography2,5-DHB 2,5-dihydroxybenzoic acid3Q Triple quadrupoleAhR Aryl hydrocarbon receptorAPCI Atmospheric pressure chemical ionizationAPI Atmospheric pressure ionizationAPPI Atmospheric pressure photoionizationC18 Octadecyl modified silicaCHCA α-cyano-4-hydroxycinnamic acidCID Collision-induced dissociationCRM Charge residue modelDa DaltonDIOS Desorption ionization on siliconEI Electron ionizationEPI Enhanced product ion ER Enhanced resolutionESI Electrospray ionizationFICZ 6-formylindolo[3,2-b]carbazoleFT-ICR Fourier transform-ion cyclotron resonanceGC Gas chromatographyGCB Graphitized carbon blackHILIC Hydrophilic interaction liquid chromatographyHPLC High performance liquid chromatographyIEM Ion evaporation modelIR InfraredIT Ion trapIXC Ion exchange chromatographyLC Liquid chromatographyLC/MS Liquid chromatography/mass spectrometryLDI Laser desorption ionization

9Abbreviations

LIT Linear ion trapLLE Liquid-liquid extractionLOD Limit of detectionMALDI Matrix-assisted laser desorption ionizationMCP Micro channel plateMDLC Multidimensional liquid chromatographyMS Mass spectrometryMSn Tandem mass spectrometryMTLI Maximum tolerable laser intensitym/z Mass to charge ratioNMR Nuclear magnetic resonanceNP Normal phaseOPE Organophosphorus triestersPSD Post source decayPTFE PolytetrafluoroethyleneQ QuadrupoleQ1 First quadrupoleq2 Second quadrupoleQ3 Third quadrupoleQA Quaternary amineRAM Restricted access materialRF Radio frequencyRP Reversed phaseSA Sinapinic acidSALDI Surface-assisted laser desorption ionizationSPE Solid phase extractionSRM Selected reaction monitoringToF Time of flightUHPLC Ultrahigh pressure liquid chromatographyUV Ultravioletv/v Volume/volumew/w Weight/weight

10 Table of contents

Table of contents

1. Introduction ..............................................................121.1 Summary of articles ....................................................14

2. Sample preparation ...............................................162.1 Solid phase extraction ................................................172.1.1 Solid phase extraction sorbents ....................................17 Restricted access material ............................................19 Graphitized carbon black ...............................................21 Modifiedsilica ................................................................232.1.2 Solid phase extraction formats ......................................242.2 Liquid-liquid extraction ...............................................26

3. Chromatographic separation .............................273.1 High performance liquid chromatography ................273.1.1 Reversed phase-high performance liquid chromatography ......................................................................................27

4. Detection by mass spectrometry ......................314.1 Ion sources ..................................................................314.1.1 Electrospray ionization ..................................................31 Matrix effects .................................................................32 Evaluation of matrix effects ...........................................33 Reduction or elimination of matrix effects .....................344.1.2 Atmospheric pressure chemical ionization ....................364.1.3 Atmospheric pressure photoionization ..........................374.1.4 Matrix-assisted laser desorption ionization ...................374.1.5 Surface-assisted laser desorption ionization .................38

11Table of contents

4.2 Mass analyzers ............................................................424.2.1 Quadrupole ....................................................................424.2.2 Ion trap ..........................................................................434.2.3 Linear ion trap ...............................................................444.2.4 Time of flight ..................................................................454.3 Detectors ......................................................................46

5. Conclusions and future perspectives .............47

6. Acknowledgments .................................................50

7. References ................................................................52

12 Introduction

1. IntroductionAnalytical chemistry refers to any kind of assessment that provides qualitative and/or quantitative information on the chemicals present in a sample. The sample in question may originate from any kind of material in any kind of location or matrix, e.g. food, an environmental compartment or living organism. Hence, the analysis is often challenging since most matrices contain not only the compound(s) of interest but also a wide range of other substances. Furthermore, the target compound(s) are often present at concentrations lower than their detection limits and require a preliminary concentration step. Therefore, the first step of an analysis is usually some kind of sample pretreatment to improve both the selectivity and sensitivity of the subsequent detection. Many techniques are available for this purpose, the suitability of which depends primarily on the physical state of the sample, e.g. solid-phase extraction (SPE) for liquid and pressurized-liquid extraction for solid samples. The types of samples analyzed during the work this thesis is based upon, and the methods chosen to apply to them, are shown in the flow chart presented in Figure 1. The next step is to detect the analyte(s) of interest. Nowadays a wide selection of instruments is available for this, and the choice depends on various factors, including the properties of the target analyte(s), their expected concentrations, the type of matrix and both the availability and costs of the instruments. Two basic approaches were adopted in the studies underlying this thesis. The first was to perform liquid chromatography/mass spectrometry (LC/MS) directly for the identification and/or quantification of the analytes. Separation of the analytes from both each other and other matrix components is very important, even after sample clean-up has been performed, since co-eluting compounds can severely interfere with mass spectrometry (MS) detection, giving false results, especially when electrospray ionization (ESI) is applied in the MS. In this approach analytes were separated using reversed phase

13Introduction

(C18-substituted particles) liquid chromatography (RPLC). In the second approach, which was designed for screening samples prior to LC/ MS in order to save time and costs, surface-assisted laser desorption ionization-mass spectrometry (SALDI-MS) was applied. SALDI is similar to matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), but superior for analyses of small molecules since the

Figure 1 Flowchart of the work presented in this thesis. After pretreatment of complex samples, the analytes of interest were identified/quantified by LC/MS either directly or after testing positive in SALDI‑MS.

Sample preparation- SPE- LLE

SALDI-ToF-MS - Screening- New surfaces

LC separation- C18 (5 µm particles)- C18 (1.8 µm particles)

MS detection- ESI- and APCI-3Q- ESI-IT- ESI-LIT

Conveyable results

Sample- Plasma- Urine- Agricultural water- Mussel

14 Introduction

matrix cluster ions in MALDI hamper the detection of peaks in the low m/z region. New surfaces were developed for more sensitive SALDI detection of target analytes. After screening, positive samples were further analyzed (qualitatively and quantitatively) by LC/MS. All the techniques mentioned here are further discussed below.

1.1 Summary of articlesPaper I describes the on-line use of a selective SPE material, RAM, for extraction and pre-concentration of organophosphorus triesters (OPEs) from human plasma prior to LC/MS. Two ionization techniques, ESI and atmospheric pressure chemical ionization (APCI), are compared in terms of the extent of observed matrix effects.

Paper II introduces graphitized carbon black (GCB) as a dual function material, capable of acting both as a SALDI surface and a SPE sorbent, for three classes of compounds: sulfonamides, human pharmaceuticals and OPEs. In the presented study a GCB µ-trap was used for off-line pretreatment, followed by SALDI-MS screening and LC/MS quantitative/structural analyses.

Paper III describes an alternative format for SPE using GCB, in which the material is incorporated in disks. Presented results show that pesticides, in both standard solutions and agricultural drainage water, can be successfully screened and quantified using a single GCB disk.

Paper IV deals with the development of a LC/MS method for identifying a metabolite of an endogenous ligand for the aryl hydrocarbon receptor (AhR), 6-formylindolo[3,2-b]carbazole (FICZ), in human urine. Enhancement of separation efficiency, by using sub-2 µm particles combined with high temperature LC, and detection sensitivity, by using a linear ion trap mass spectrometer (LIT-MS), is demonstrated.

15Introduction

Paper V reports the preparation of an acidic SALDI surface, oxidized GCB. Titration, nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscopy data presented demonstrate that the surfaces of oxidized GCB nanoparticles possess more carboxylic groups than GCB. Propranolol was extracted from Baltic Sea blue mussels and quantitatively determined utilizing oxidized GCB.

Another new SALDI surface, formed from silicon nitride nanoparticles, which is suitable for the analyses of small molecule drugs is presented in Paper VI. Its potential use in analyses of drugs in real matrices such as urine is also illustrated.

16 Sample preparation

2. Sample preparationSample preparation is an essential part, and often the time-determining step, of an analytical scheme. It significantly affects the quality of the results obtained, throughput and costs of analyses. Thus, selecting an appropriate sample preparation method, and optimizing it, are key steps in the development of an accurate, reliable analytical procedure.No universal sample preparation techniques suitable for all kinds of samples have been developed, but diverse methods are routinely and successfully applied to various types of samples, such as liquid-liquid extraction (LLE), SPE and supercritical fluid extraction [1, 2]. The most appropriate sample preparation method for a given application is dependent on the nature of the analytes, sample matrix, separation method and the type of detection technique to be used. In the work presented in this thesis a selection of analytes, such as OPEs, sulfonamides and pesticides were extracted from a variety of matrices, including: plasma (Paper I), urine (Papers II, IV and VI), environmental water samples (Paper III) and mussels (Paper V). In all cases analytes were separated by high performance liquid chromatography (HPLC) and if no sample pretreatment had been performed the column may have become blocked, and in extreme cases irreversible adsorption of substances to the column may have occurred. The ionization techniques used in the subsequent MS detection were APCI, ESI and SALDI, which often require sample preparation to provide sufficiently clear signals by removing interfering components and increasing the concentration of analytes to levels exceeding the limits of detection of the technique. SPE, with a variety of sorbents in different formats, as well as LLE, were used during the studies, and are further discussed below.

17Sample preparation

2.1 Solid phase extractionSolid phase extraction (SPE) is a widely used sample preparation technique in which selected analytes are isolated from a sample and retained on a solid phase, then recovered (eluted) from it while interferents either pass straight through the column or remain adsorbed to it when the analytes are eluted. The main goals of SPE are to clean-up samples, selectively enrich analytes and/or change the medium. The SPE process usually consists of four steps. Firstly, conditioning of the sorbent to promote good surface contact between the sample and sorbent. Secondly, passing of the sample through the sorbent bed by gravity or application of vacuum or pressure. Thirdly, washing away retained interferents while avoiding elution of analytes. Fourthly, elution of the analytes retained on the sorbent using a suitable solvent or mixture of solvents.

2.1.1 Solid phase extraction sorbentsSelecting an appropriate SPE sorbent is critical for efficient recovery of analytes from a liquid sample. An effective SPE sorbent must be able to absorb high and reproducible proportions of the analytes and allow their complete elution. In other words the sorption process must be reversible. In addition, SPE sorbents should be selective towards the analytes of interest, have large surface areas, exhibit good surface contact with the sample solution, be stable in the sample matrix and the solvents used and be free of leachable impurities [3].The most suitable type of SPE sorbent to use for a given application is primarily determined by the sample solvent composition followed by the main mechanism whereby the analyte(s) of interest interact with it, Figure 2 [4].


Figure 2 Schematic outline of SPE selection based primarily on the physico‑chemical nature of the sample matrix [4].

SPE is a widely used technique because it is suitable for both non-polar and polar compounds. A large range of sorbents are available for this purpose, ranging from non-polar adsorbents, such as C18-substituted phases, to ion exchange materials and from widely applicable ones to compound-specific adsorbents such as molecularly imprinted polymers [5] and RAMs (Paper I). The most widely used commercially available SPE sorbents are bonded-phase silica particles with chemically modified siloxane groups, such as C18 (which were used in Study IV). Sorbents such as GCB (Papers II and III) and porous graphitic carbon [6] are examples of carbonaceous sorbents that can be used for trapping polar compounds from diverse matrices such as sediment samples [7], water [8] and food [9].

Normal phase (e.g. CN, NH2) Low polarity

High polarity

Organic sample

Ionized

Neutral

Reversed phase or Ion exchange

Reversed phase or Normal phase

Reversed phase (e.g. C18, C8)

Strong cation exchange (e.g. SO3

-) or Weak cation exchange (e.g. COO-)

Anionic

Cationic

Neutral

Ionized

Strong anion exchange (e.g. NH4

+)

or Amino (e.g. NH3

+)

Aqueous sample


Restricted access materials Restricted access materials (RAMs) allow the direct and multiple extraction and enrichment of compounds in untreated fluids such as plasma. Plasma is a complex matrix consisting of about 92% water and proteins such as albumin, as well as diverse smaller compounds. To determine a target analyte in plasma it first has to be separated from proteins, which was done using a RAM in Study I. Macromolecules such as proteins are prevented from reaching the bonded phase, which is present on the internal surfaces of porous particles in a RAM, by a polymer network at the outer surface of the particles or by pore diameter restrictions. Simultaneously with this size-exclusion process, low-molecular weight analytes are selectively retained by other chromatographic interactions, such as reversed phase (RP) [10], ion exchange [11] or more specific, affinity binding [12].The RAM used in Study I, LiChrospher® ADS, alkyl diol silica (Merck, Germany), had a pore diameter of approximately 60 Å, which blocked the penetration of macromolecules with molecular weights greater than 15 000 Da [13]. The external surface of the spherical particles was bound to hydrophilic and electroneutral diol groups, protecting the sorbent from contamination by proteins and allowing multiple injections of plasma. The internal surface of the porous particles had a hydrophobic bonded phase (C18 alkyl chains), freely accessible to the target analytes (OPEs), Figure 3. OPEs are mostly employed as flame retardants and/or plasticizers, depending on the desired properties, in a variety of products [14]. They are mainly used as additives, i.e. they are not usually chemically bound to the products they are added to, and therefore may diffuse into the environment. Their occurrence has been reported in both indoor environments, e.g. offices, schools, day care centers [15] and outdoor environments, e.g. river water [16], groundwater [17], wastewater [18], drinking water [19], soil [20], sediment [21] and pine needles [22]. The presence of OPEs in humans


has also been studied, but to a lesser extent, and tris(dichloropropyl) phosphate has been found in seminal plasma [23] while tributoxyethyl phosphate and tris(1,3-dichloropropyl) phosphate in adipose tissue [24]. OPEs in various matrices are usually determined by a clean-up and extraction/enrichment step followed by gas chromatography (GC) or LC/MS analysis. There are far fewer published LC/MS methods for their determination than GC-based techniques, but LC/MS offers possibilities to determine more polar compounds, such as diesters and monoesters, as well as direct injection of aqueous samples without the need for analyte transfer into an organic solvent [25]. The method described in Paper I for the determination of nine trialkyl and triaryl phosphates in human plasma was the first published LC/MS method [25], although several GC-based methods had been previously presented [26-28].

Figure 3 Schematic sketch [29] (with permission from Elsevier) (a) and diagram of LiChrospher® ADS material (b). R groups may be C4 , C8 or C18 groups [13] (with permission from Elsevier).

Pre-columns filled with RAM are suitable tools for on-line injection of samples using column switching, as demonstrated in Paper I. On-line sample preparation prior to chromatography offers several advantages over the off-line approach. In most cases the throughput is increased, it provides higher sensitivity since the whole extract is transferred to the analytical system and problems associated with sample

(a) (b)

Pore diameter: 60 Å

OH

OH

OH

OH

OHOH

O

C OR

O

C OR

In Out


contamination or degradation are avoided. However, on-line systems also have disadvantages, especially their complexity (which makes their optimization more demanding) and the limited commercial availability of instruments [30].When comparing the performance of RAM sorbents with common SPE sorbents, a major factor is the lifetime. Even though the price of a RAM pre-column is usually high, its much longer lifetime compared to SPE cartridges, that are designed for a single use, makes it a good alternative. Almost 100% of the protein that is injected into a RAM column is eluted and the on-line coupling of RAM prevents the loss of analytes during pretreatment, as well making it a safer way of handling hazardous and infectious samples [31].

Graphitized carbon black The use of carbon-based sorbents for SPE began in the 1980s with the introduction of graphitized carbon blacks (GCBs) [32]. GCBs are obtained from heating carbon black in an inert atmosphere at 2700-3000ºC. The heterogeneous surface of carbon black is covered with a layer of chemisorbed oxygen, which is not part of the surface structure of carbon black but is bound to large polycyclic molecules that are strongly adsorbed to the carbon surface. Heating carbon black results in the removal of most of the polycyclic molecules, and thus the chemisorbed oxygen from the surface, as well as forming a graphitic structure via the rearrangement of carbon atoms [33]. Interest in using GCBs as SPE sorbents increased when their ability for isolating polar molecules, which have low affinity for most RP sorbents, was demonstrated. Compounds are retained on GCB as a result of the presence of two types of adsorption sites [34]. The most abundant sites are provided by the non-polar carbon atoms arranged in a graphite-like structure, which interact with compounds through van der Waals forces. However, GCB also has anion exchange properties due to the presence of


positively charged chemical impurities on its surface. More specifically, oxygen complexes with a chromene-like structure, originating from the heating of carbon black, rearrange to benzpyrylium salts in the presence of acidified water and are responsible for binding to anions via electrostatic forces [33]. As a consequence of the anion-exchange properties basic and neutral compounds can be separated from acidic compounds using GCB and suitable eluant mixtures [8]. It is even possible to sub-fractionate acidic compounds according to their pKa values by including appropriate additives in the elution mixture [35].In Studies II and III, in which GCB was used as a solid-phase extraction medium, excess breakthrough of some compounds was prevented by washing GCB with acetic acid prior to passing samples through it. On the surface of GCB there are also hydroquinone groups, which are in equilibrium with their oxidized forms, namely semiquinones and quinones [33]. Particular compounds can bind to the surface of GCB via quinone chemisorption, which makes their adsorption partially irreversible and results in low recoveries [8]. This problem was overcome by passing an ascorbic acid solution through GCB and reducing the quinone groups to less reactive hydroquinones. After samples had been passed through the GCB the material was rinsed with methanol to eliminate any residual water that was not removed by vacuum, since the presence of water considerably reduces the elution efficiency of the hydrophobic solvent mixture, by hindering contact between the desorption mixture and GCB [8]. In initial experiments 80:20 volume/volume (v/v) CH2Cl2/MeOH was then used to elute analytes (Paper II), based on findings by Di Corcia and Marchetti [8] that neither dichloromethane nor methanol alone is able to elute some of the compounds and the 80:20 v/v mixture provides the best elution recoveries. However, methanol is reportedly able to displace the analytes that are adsorbed on the active centers of GCB, via hydrogen bonding, and the replacement of methanol by acetonitrile results in


lower recoveries for such compounds [8]. Nevertheless, the recoveries of acidic analytes (naproxen and diclofenac, Paper II) were low when this elution mixture was acidic, so a basic mixture of CH2Cl2/MeOH (80:20 v/v) containing 50 mM NaOH was used, which resulted in almost full recoveries of these substances.An important advantage of GCB, especially when dealing with large volumes of environmental samples, is its storage stability. It has been shown that problems associated with sample alteration during the period between sampling and analysis can be solved, and storage space saved, by storing analytes of interest on GCB [36, 37].

ModifiedsilicaThe first article regarding SPE using commercially available bonded-phase silica (C18 phase) was published in 1978 [38] and this is still the most commonly used type of solid phase for SPE. Bonded stationary phases are prepared by attaching organic polar, non-polar or ionic ligands to silica particles via reaction with the silanol groups. C18 is the most widely used RP sorbent for adsorbing analytes from aqueous samples and was used in Study IV to extract and concentrate metabolites of 6-formylindolo[3,2-b]carbazole, FICZ, from human urine. In this study the aim was to demonstrate the presence of FICZ, a proposed ligand for the AhR formed by exposure of tryptophan to light, in the human body by detecting its stable metabolites in urine. Metabolites are generally formed by introducing a functional group such as –OH, -NH, -SH or –COOH into a molecule, increasing its hydrophilicity. However, a further increase in the hydrophilicity is required to promote excretion of the substance via urine and this is achieved through conjugation with species such as sulfonate and glucuronide. Figure 4 illustrates the several steps involved in the formation of the targeted conjugated FICZ metabolite in urine.


Figure 4 The formation of 8‑SO4-FICZ, identified in human urine by SPE/LC/MS/MS. In the presence of light tryptophan produces FICZ, which is metabolized in the body into a number of compounds including 8‑OH‑FICZ. 8‑OH‑FICZ is then conjugated with a sulfonate group generating 8‑SO4‑FICZ.

It is easy to collect urine in large volumes, but the concentrations of its constituents can vary substantially depending (inter alia) on the volume of liquid that is consumed by the subject. Its main constituents (apart from water) are urea and salts, such as sodium chloride. Therefore, to account for variations in concentrations the density of all urine samples used was measured. In addition to those mentioned, thousands of other compounds (mainly polar molecules, e.g. small organic acids) are usually present in urine, but most of the constituents are removed when using a non-polar SPE sorbent such as a C18 phase.

2.1.2 Solid phase extraction formatsThe extraction medium used in SPE can often be applied in various formats, such as cartridges, syringes and disks [39]. The preferred format depends on the availability as well as the application. Cartridges and syringes can be prepared in the laboratory while disks are produced by manufacturing plants, thus a more limited range of sorbents is available in disks. In the studies this thesis is based upon SPE was applied in a variety of formats. Commercially available RAM and Sep-Pak® cartridges were used in Studies I and IV, respectively. SPE µ-traps were constructed

hydroxylation sulfonationNH

NH

OOH

NH

NH

OO

SO

O OH

NH

NH2

OOH light

Tryptophan FICZ

NH

NH

O

8-OH-FICZ 8-SO4-FICZ


in-house by filling pipette tips with GCB particles in Study II. In Study III disks were used that were manufactured by 3M as sheets, using EmporeTM disk technology [40], consisting of 10% weight/weight (w/w) GCB particles in an inert network of polytetrafluoroethylene (PTFE) fibrils (90% w/w). The sheet was cut into 13 mm disks (Figure 5a) and each disk was sandwiched between two Swinnex® gaskets (Figure 5b) and placed in a Swinnex® filter holder (Figure 5c).

Figure 5 A sheet of GCB cut into 13 mm disks (a), a disk to be sandwiched between two Swinnex® gaskets (b) and a Swinnex® filter holder (c).

The same types of particles are used in disks, cartridges and syringes, except that smaller particles are used in disks than in conventional cartridges, providing two major advantages: smaller elution volumes and higher flow rates. The smaller particle size results in an increase in the surface area and reduces the void volume, promoting partitioning and therefore less sorbent is required to process a certain volume of sample, which in turn results in lower solvent volume requirements for elution. The smaller particle size also allows more dense and uniform packing, reducing breakthrough and channeling and permitting the use of higher flow rates [41].

(a) (c)(b)


The diverse formats of SPE devices provide opportunities to employ them on-line or off-line prior to use of analytical systems such as LC/MS instruments, and to automate the whole process. All the SPE analyses in the studies underlying this thesis were performed off-line except for the on-line RAM analyses described in Paper I. GCB disks (Paper III) could easily be used in a 96-well format, allowing automation of the entire SPE/SALDI/LC/MS procedure.

2.2 Liquid-liquid extractionOne of the most versatile techniques for extraction and enrichment of analytes from a liquid sample is liquid-liquid extraction (LLE), which is based on the distribution of an analyte between two immiscible solvents, one aqueous and one organic. After the solvents are mixed two phases are formed and the analyte partitions largely into the solvent it is more soluble in. This technique was applied in Study V to extract propranolol from Baltic Sea blue mussels, part of an ecotoxicological study. LLE has several drawbacks compared to SPE, e.g. it is less environmentally friendly and safe due to the relatively high consumption of solvents, but improvements in automation of LLE have resulted in its common use prior to MS [42]. Automation of LLE has been facilitated by the introduction of chemically inert solid supports, such as EXtrelut® NT (Merck) and Hydromatrix (Varian). Aqueous samples become distributed as thin films when applied to these supports, and hydrophobic compounds can be subsequently eluted using an organic solvent that is immiscible with water, while the aqueous phase (and hydrophilic compounds) remain on the solid support.

27Chromatographic separation

3. Chromatographic separationAfter sample clean-up there is still a need for chromatographic separation of the components present in samples prior to detection, especially when dealing with complex matrices such as urine and plasma, in which some matrix components may still be present even after sample pretreatment. Separating the matrix components from the analytes of interest assists detection of the latter, especially when performing ESI-MS, as discussed in section 4.1.1. HPLC was the separation technique used in all of the studies this thesis is based upon, and hence it is further discussed below.

3.1 High performance liquid chromatographyHPLC is a technique in which dissolved compounds in a liquid medium (the mobile phase) are separated according to their partitioning between the mobile phase and a stationary (solid) phase. Stationary phases in which organic moieties are joined to a silica surface are the most widely used, but diverse stationary phases based on a variety of separation modes such as RP, normal phase (NP), hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IXC) are available. NP and HILIC stationary phases are suitable for retention of polar compounds, however it is possible to use a mixed organic-aqueous mobile phase with HILIC phases, instead of a non-polar mobile phase with NP phases, making the former compatible with ESI-MS [43]. IXC is another option for separating polar compounds, however to be able to couple it to MS the use of nonvolatile inorganic salts for elution should be avoided. RPLC can effectively separate non-polar compounds, and was the choice in Studies I, II, III and IV.

3.1.1 Reversed phase-high performance liquid chromatographyRP-HPLC involves the use of a non-polar stationary phase and a polar mobile phase for separating compounds based on their hydrophobicity. It is often compatible with ESI-MS since a high fraction of the mobile phase

28 Chromatographic separation

is organic. A variety of stationary phases are available for RP-HPLC, but the most commonly used are C18 derivatives. When RP-HPLC was used in the studies reported here, endcapped C18 columns with minimized secondary interactions from residual silanol groups were applied.Substantial improvements in column efficiency have been enabled by the recent development of “ultrahigh pressure liquid chromatography (UHPLC)” [44] instruments capable of handling pressures as high as 1200 bar, which has enabled the use of stationary phases with smaller than previously possible particles (sub-2 µm). The possibility of connecting UHPLC systems to MS detectors makes it an even more powerful and attractive technique. However, due to the very narrow peaks produced by UHPLC, they can only be coupled to MS instruments with high acquisition rates, e.g. ToF instruments [45]. In Study IV a column filled with 1.8 µm C18 particles was used to enhance the separation and sensitivity of detecting monosulfated-FICZ metabolites in human urine since it was not possible to detect them using conventional size C18 particles. However, when this work was performed UHPLC instrumentation was not accessible so high temperatures were used to decrease the mobile phase viscosity and hence allow the use of higher flow rates. The column was placed in a GC-oven at 80° C and the mobile phase was pre-heated. It should be noted when using high temperature HPLC one has to consider the other effects that high temperature also has on RP-HPLC, such as increases in mass transfer, reduction of eluent strength and changes in selectivity [46]. The improvement observed in the separation efficiency is demonstrated by the UV chromatograms shown in Figure 6 (unpublished data), obtained from two portions of a pre-treated urine sample: one injected onto a C18 column with 5 µm particles (a) and the other injected onto a C18 column with 1.8 µm particles (b).

29Chromatographic separation

Figure 6 UV chromatograms of two portions of a urine sample, one separated on a C18 column with 5 µm particles at room temperature (a) and the other on a C18 column with 1.8 µm particles at 80 °C (b).

To improve the resolution of most separations an option is to use multidimensional liquid chromatography (MDLC). All the LC done in the studies this thesis is based upon was one-dimensional (1DLC), but use of MDLC would have been beneficial in Study IV since many overlapping metabolites were detected in the urine samples, despite using a column with 1.8 µm particles and high temperature. MDLC allows the combination of two or more independent or nearly independent separation steps, thereby increasing the peak capacity of the corresponding 1DLC techniques and thus significantly improving

30 Chromatographic separation

the separation of compounds in complex samples [47]. MDLC can be divided into three categories: off line MDLC [48], on-line MDLC [49] and comprehensive LC (LC × LC) [50, 51]. In off-line MDLC, the fractions of interest from the first LC column are manually collected, evaporated and injected into the second LC column. In on-line MDLC, the two separation systems are connected, and selected fractions from the first LC are automatically transferred to the second LC, but as in off-line MDLC, only the fractions of interest are re-injected into the second dimension column. In LC × LC, the whole eluate from the first LC is continuously collected and injected into the second dimension column for separation. Another alternative to enhance the efficiency of a separation that can be applied either with or without UHPLC equipment, is to use columns filled with core-shell particles such as Kinetex-C18 [52] or Halo-C18 [53]. Such particles are prepared by growing a homogenous porous shell on a solid silica core. They reduce the Eddy diffusion effects due to their extremely narrow particle size distribution and increase the mass transfer speed since the particles are not fully porous so analytes spend less time diffusing in and out of the pores.

31Detection by mass spectrometry

4. Detection by mass spectrometryMass spectrometers are powerful detectors that are capable of ionizing and fragmenting many classes of compounds in various ways, separating them in gas phase according to their mass to charge ratio (m/z), and finally detecting them. MS instruments consist of three major sections: an ion source that produces gas phase ions, an analyzer that separates ions according to their m/z ratios and a detector that counts the ions. Each of these parts is briefly discussed below, focusing on the types used in the studies this thesis is based upon.

4.1 Ion sourcesNowadays a number of atmospheric pressure ionization (API) sources, such as ESI [54], APCI [55] and atmospheric pressure photoionization (APPI) [56] are available for LC/MS instruments, with ESI being the most common API interface used in analyses of biological and environmental samples. In API the ionization occurs at atmospheric pressure and it is a ‘soft’ ionization mode, since the predominant ion detected is the quasimolecular ion rather than other ions resulting from fragmentation of the molecule within the source. An alternative ionization technique, performed at low pressure, is electron ionization (EI). LC/EI-MS spectra obtained for small molecules are comparable to GC/EI-MS spectra, and commercially available EI-MS instruments provide full searching capacity [57]. APCI was used in Study I and ESI in Studies I, II, III and IV, hence these modes are briefly described below.

4.1.1 Electrospray ionizationIn LC/ESI-MS an electrical field, under atmospheric pressure, is generated by applying a potential difference between the needle that the liquid from the LC system passes through and the entrance to the mass analyzer. This electrical field results in the formation of a spray of charged droplets, which is assisted by a flow of a neutral gas, e.g.

32 Detection by mass spectrometry

nitrogen. A common practice is to heat the ESI needle as well as the neutral gas and to use cone voltage to assist evaporation of neutral solvent molecules in the droplets. Very small charged droplets are then produced when the charge repulsion is sufficient to overcome the surface tension and consequently a Coulombic explosion occurs. The mechanism whereby gas-phase ions are produced from the very small droplets is not yet fully understood, but two main models have been postulated: the charge residue model (CRM) and the ion evaporation model (IEM). According to the CRM, gas-phase ions are products of the evaporations and fissions, which result in single ions, while the IEM assumes the emission of ions from the small droplets into the gas phase [58]. According to both models, the analytes in ESI are ionized in solution. The formation of adducts is a common phenomenon in ESI. However, it is possible to increase or decrease the amount of adducts formed by altering the mobile phase solvent composition [59, 60]. A drawback of ESI is its sensitivity to the sample matrix, as described below, which led to the use of another ionization source in Study I, namely APCI.

Matrix effects Matrix effects can be described as the differences between mass spectrometric responses to analytes in a standard solution and the same analytes in a different matrix, such as plasma or a sediment extract. Matrix effects are due to matrix constituents (or exogenous contaminants, e.g. plasticizers from containers used to store samples [61]) that interfere with the ionization of analytes by co-eluting, resulting in either ion suppression or ion enhancement with significant potential losses of accuracy and precision.The mechanism of matrix effects, in both ESI and APCI, has been the subject of many studies. It has been suggested that matrix effects in ESI are primarily due to liquid phase processes that occur as a result of high


concentrations of nonvolatile constituents in the sample or nonvolatile mobile phase additives, which prevent the analyte ions from escaping from the droplet into the gas phase [62]. Other proposed mechanisms are based on the effects of matrix components on surface activity in the ESI process [63]. The term ‘surface activity’ in this context refers to the affinity of an analyte for the air-liquid interface of ESI droplets. Matrix components with higher surface activity than an analyte may suppress the ionization of the analyte by competing for access to the droplet surface and (hence) gas-phase emission [63]. A possible cause of matrix effects in APCI could be the formation of solid components via the co-precipitation of analytes and nonvolatile sample components [62]. APCI is known to be less susceptible to matrix effects than ESI [64], as found in Study I, however both ion suppression [65] and enhancement [66] have been observed when using APCI.The observed differences in ion suppression/enhancement between APCI and ESI are probably due to their different mechanisms of ionization (ion evaporation from the liquid droplet in ESI versus gas phase chemical ionization in APCI) [62].

Evaluation of matrix effectsThere are two common ways to evaluate matrix effects. One is the post-column infusion method, in which an analyte is constantly infused into an extract of a sample after the chromatographic column while the instrument response is being monitored (Figure 7a). This method provides a qualitative assessment of matrix effects, showing the chromatographic regions most prone to matrix effects, but does not provide any quantitative measurements of matrix effects. The second method is the post-extraction spike method, in which the response to an analyte in a standard solution is compared to the response to the same analyte spiked into a blank matrix sample after it has been through the sample preparation step (Figure 7b), hence it provides quantitative


evaluations of matrix effects [67]. Both of these approaches were applied in Study I. The matrix effects were quantified in both ESI and APCI using the post-extraction spike method and further evaluated by post-column infusion.

Figure 7 Schematic presentation of the procedures performed for evaluating matrix effects. In the post‑column infusion method a standard solution of analytes was infused into extracted blank plasma after the HPLC column and before the mass spectrometer (a). In the post‑extraction spike method a standard solution of analytes was injected into extracted blank plasma before entering the HPLC column (b).

Reduction or elimination of matrix effectsTo reduce or eliminate matrix effects in quantitative analysis two general approaches can be adopted. Firstly, sample components responsible for matrix effects can be removed by improving the sample pretreatment and/or chromatographic separation. Secondly, the influence of matrix effects on the accuracy/precision of the method can be minimized by optimizing the ionization method, mobile phase composition or flow rate and LC/MS instrumentation [68].Improvements in sample preparation can be achieved by using more selective SPE materials as presented in Paper I, use of RAM, and in Papers II and III, use of GCB. Alternative approaches for reducing the

RAM Column HPLC Column

Syringe pump

Mass spectrometerT-coupling

Standard solution

(a)

Blank plasma Standard solution

(b)

Injection valve


amounts of matrix components reaching the analytical system include sample dilution [69] and use of smaller injection volumes [70]. However, both of these measures increase the detection limits of the compounds of interest, which may make it difficult to detect them. A change in the ionization mode, for example from ESI to APCI, APPI or EI [71] may also reduce or eliminate matrix effects, as observed in Study I, which led to the use of APCI as the preferred ionization technique. However, this may not be possible for all target analytes because some do not give responses in APCI-, APPI- or EI-MS. Another way to improve the correlation of LC/MS signals between analytes in sample matrices and standards (i.e. reduce matrix effects) is to change the composition of the mobile phase by, for example, using appropriate buffers or additives [70]. Reduction of the LC flow rate, either in conventional ESI [72] or using nanoelectrospray [73], has also proven to reduce matrix effects since less matrix components are introduced into the ionization source during any given period of time. Matrix effects also depend on the design of the ionization source, as well as the presence of exogenous compounds originating from, for example, plastic materials used in sample storage containers. Therefore using a LC/MS instrument from another manufacturer and a different brand of plastic material may change the scale of these effects [74].The most effective way to compensate for matrix effects is probably the standard addition method. However, this is time consuming, laborious and (hence) not frequently used [75]. Other approaches include use of isotopically-labeled or (as in Study I) structurally similar compounds as internal standards. However, the high cost and limited availability of isotopically-labeled compounds restrict their use, and even the slight difference in the retention times of an analyte and isotopically-labeled standard can result in some degree of signal alteration [76]. When using either of these kinds of internal standards the best results are achieved when the compounds of interest and standards elute close to one another


and are affected by the same matrix components. Consequently, in a multiresidue analysis the introduction of several internal standards is required, ideally one per analyte. A third option is calibration using matrix-matched standards. However, this requires the availability of a control matrix that is comparable to the sample matrix and does not contain the target compounds. A fourth possibility is the echo-peak technique in which the unknown sample and the standard solution are injected consecutively within a short time period in each chromatographic run, so that the standards should be subject to very similar matrix interferences. However, this approach does not adequately correct for matrix effects if the peak profiles of interfering matrix components are very sharp and do not coelute with both the analyte and its echo-peak, or if the analyte gives a highly tailing peak [77].

4.1.2 Atmospheric pressure chemical ionizationIn atmospheric pressure chemical ionization (APCI) analytes are chemically ionized at atmospheric pressure using nitrogen and water vapor as reagent gas and electrons from the corona discharge needle, which is situated between the spray chamber and the orifice through which samples are introduced into the mass spectrometer. High temperatures are used to assist the evaporation of the liquid entering the mass spectrometer from the LC, therefore it can only be used for relatively thermo-stable compounds, and since the ionization occurs in gas phase, only volatile analytes are ionized [78]. APCI has the advantages of being capable of handling higher flow rates from separation systems, ionizing less polar compounds and being less prone to matrix effects, as discussed in the previous section, compared to ESI. In Study I APCI was chosen over ESI for the ionization of the OPEs, although ESI gave lower limit of detection (LOD) values, due to the less severe matrix effects observed using APCI.


4.1.3 Atmospheric pressure photoionizationAtmospheric pressure photoionization (APPI) is similar to APCI in that in both techniques ionization of the analytes takes place in the gas phase, but in APPI a modified APCI source containing a heated nebulizer is used. Instead of a corona discharge needle a krypton vacuum ultraviolet (UV) lamp is employed as a source of photons to ionize the analytes present in the vapor phase [56]. APPI-MS has been applied in the determination of a variety of compounds, e.g. compounds of environmental concern such as brominated flame retardants [79] and polycyclic aromatic hydrocarbons [80], drug metabolites [81] and explosives [82].

4.1.4 Matrix-assisted laser desorption ionizationMatrix-assisted laser desorption ionization (MALDI) was introduced in 1988 by Karas and Hillenkamp [83, 84] and Tanaka [85]. In MALDI, the compound to be determined is dissolved in a solution containing matrix molecules that strongly absorb at the laser wavelength used. Various kinds of lasers providing light with different specific wavelengths can be used, such as CO2 lasers (10.6 µm) or nitrogen lasers (337 nm) [86]. Various matrices can also be used; common UV-MALDI matrices include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB) and sinapinic acid (SA), while matrices used with IR-MALDI include urea, carboxylic acids, alcohols and water. In each case, the analyte-matrix mixture is dried on a metal plate (co-crystallized) before analysis and then placed in the source of the MALDI instrument, which can be either at atmospheric pressure or under vacuum. The spots are then irradiated by laser pulses for a short duration. The mechanisms of desorption [87] and ionization [88, 89] in MALDI are not completely understood. However, the laser irradiation induces rapid heating of the matrix crystals, which causes the expansion of the matrix into gas phase, taking the intact analyte molecules to the matrix plume [87]. The ion formation mechanism involves proton transfer in the liquid phase before


desorption, or in the gas-phase from photo-ionized matrix molecules. The ions in the gas phase are then accelerated towards the analyzer. In positive MALDI sodium and potassium adducts of analytes are frequently detected, as well as their proton adducts [89].MALDI provides soft ionization of a wide range of analytes, inter alia carbohydrates [90], synthetic polymers [91], intact microorganisms [92], proteins and protein complexes [93] and small molecules [94]. Mild ionization conditions, high sensitivity, rapidity and high tolerance to contamination by detergents, salts, buffers and other potential interferents are the main advantages [95], which make the technique more suitable for some applications than ESI. However, conventional MALDI is not as suitable for detecting small (<500 Da) molecules due to high background noise from fragment and cluster ions of common organic matrices. Therefore, other LDI (laser desorption ionization) techniques such as SALDI have been developed in recent years and were also investigated in Studies II, III, V and VI.

4.1.5 Surface-assisted laser desorption ionizationMany approaches and materials have been used in attempts to overcome the matrix problems associated with the determination of small molecules such as: desorption ionization on silicon (DIOS) [96], polymeric matrices [97], inorganic materials [98], high molecular weight matrices [99], surfactant-suppressed matrices [100] and carbon allotropes. Carbonaceous materials such as carbon nanotubes [101], activated carbon [102], graphite [103] and colloidal graphite [104] have been shown to provide suitable surfaces for surface-assisted laser desorption ionization (SALDI) and have been used in various formats for different applications. In Study II a carbon-based material, GCB, was introduced for the first time as a SALDI surface. It was found that the maximum tolerable laser intensity (MTLI) at which GCB provided a clean background spectrum without any interference from cluster ions was


higher than that of activated carbon. This feature is advantageous in the determination of small molecules since most of the interferences appear in the low mass range, as well as when performing post source decay (PSD) analysis. The resolution and mass accuracy obtained using GCB was compared to corresponding parameters for 2,5-DHB and DIOS. The mass accuracies obtained from the three media were not significantly different, and the mass resolution attained using GCB was intermediate between that attained with 2,5-DHB and DIOS. In Study III GCB disks, consisting of GCB particles loaded in a PTFE network, were used for the SALDI analysis of a selection of pesticides. Again, a clean background free from GCB and PTFE interferences was achieved. However, handling the GCB, e.g. placing it in the LDI instrument, was more convenient in the disk format than in the powder format.The capability of GCB to provide a good SALDI surface, as well as being a SPE sorbent, enabled the integration of these properties and development of a convenient sample screening procedure in Studies II and III. Increasing the throughput of analyses by relatively fast screening and elimination of negative samples is valuable, especially when dealing with large numbers of samples, as in many environmental studies for instance. In Study II GCB particles were packed in a µ-trap and after each sample had passed through it a small portion of the particles was removed and placed on a stainless steel plate for SALDI-MS analysis. Positive samples were identified in this fashion and the negative samples were discarded. The analyte(s) retained on the µ-tips giving positive results were then desorbed using appropriate solvents and further identified and quantified by LC/MS. A similar approach was adopted in Study III, but the GCB was used in disk rather than µ-trap format, since disks offered higher SPE efficiency, as well as a more appropriate format for LDI instruments. When using this procedure, one must note


the LODs of the compounds of interest in the SALDI spectra since it is the sample elimination factor. To reduce detection limits in SALDI, the desorption ionization efficiency was enhanced by increasing the number of cation-exchangeable sites on the surface of GCB and modifying the desorption process through oxidation (Paper V). This was done using a concentrated H2SO4/HNO3 mixture, which generated surface oxide sites while reducing the size of the GCB particles to nano-dimensions. SEM elemental analysis revealed the oxygen content of GCB to be 5.5 ± 0.1% (w/w), while oxidized GCB contained 14.1 ± 1.3% (w/w) oxygen. The nature of the oxygenated functional groups was studied by acid-base titration and NMR, and it was concluded that approximately 5% (w/w) of the oxygen in oxidized GCB is in functional groups other than COOH. The structures of a number of possible acidic surface oxides are shown in Figure 8 [105].

Figure 8 A number of possible structures of surface oxides in carbon blacks [105].

The oxidation of GCB was also demonstrated by comparing suspensions of GCB in water before and after oxidation. GCB and oxidized GCB were dispersed separately in water with shaking, then rested at room temperature. As shown in Figure 9, GCB particles all sedimented to the bottom of the container within a few minutes of resting due to their hydrophobic surface, whereas the oxidized GCB particles remained well dispersed in water, forming a stable suspension. Since the extent to which particles remain suspended in water depends on their size and amount of hydrophilic groups, the nanoparticles (illustrated in SEM

OO O O O OOOH


images of Paper V, Figure 1) have more hydrophilic groups attached to their surface as a result of the oxidation procedure.

Figure 9 GCB (left) and oxidized GCB (right) in water.

Differences in desorption profiles between GCB and oxidized GCB were evaluated using quaternary amines (QAs) with different alkyl chain lengths as model compounds. Relatively less hydrophobic QAs were more easily desorbed from GCB, while more hydrophobic QAs were more readily desorbed from oxidized GCB. These profiles also suggest that the hydrophobic surface of GCB is made more hydrophilic by oxidation, and that the improvement in signal to noise ratios obtained using oxidized GCB is due to both improvement in the ionization of the target compounds and changes in the desorption process.Nanoparticles and nanostructures, such as mesoporous tungsten and titanium dioxide [106] and silicon nanowires [107] are another group of compounds used in LDI-MS analysis of small molecules. A potential advantage that metal nanostructures offer over chemical matrices is that their absorption is nearly independent of the wavelength used, whereas the absorption of chemical matrices is restricted to their absorption bands [108]. In the study described in Paper VI silicon nitride nanoparticles were used as a SALDI surface for determination of small molecule drugs from water and spiked urine.


4.2 Mass analyzersAfter the ion source, the ions present in the gas phase are transferred to the mass analyzer, where they can be separated according to their m/z ratios. Several types of mass analyzers are commercially available: quadrupole (Q), ion trap (IT), linear ion trap (LIT), time-of-flight (ToF), Fourier transform-ion cyclotron resonance (FT-ICR) and orbitrap [109, 110]. In an FT-ICR instrument ions are trapped and detected in a single cell placed within a superconducting magnet. Such instruments have extremely high mass resolution and accuracy. The orbitrap, a relatively new type of mass analyzer, uses an electrostatic field to trap stable ions orbiting around an axial electrode. Its advantages include high ion capacity, high mass resolution and accuracy, wide dynamic range and broad mass range [111]. The other analyzers mentioned above were used in the studies underlying this thesis and are discussed below.

4.2.1 Quadrupole Quadrupole (Q) mass analyzers are the most common type of analyzer found in modern analytical laboratories. However, Qs are used not only in mass analyzers but also in ion transfer optics, collision cells and LITs. A Q consists of four cylindrical rods to which a fixed direct current (DC) and an alternating radio frequency (RF) are applied, creating a Q field that only allows ions within a certain m/z range to pass through them. The ions outside that m/z region hit the rods and discharge. The resolving power of Qs is usually one throughout their functional mass range, which is often up to m/z 4000. Single Q instruments have limited selectivity and their typical applications, when hyphenated with HPLC, include confirmation of molecular weights of target analytes and automated preparative HPLC fractionations. However, selectivity can be increased by using “in-source CID (collision-induced dissociation)”. In this process ions are activated in the region between the API source and the analyzer,


leading to fragmentation, but since no precursor ion is selected the fragment ions cannot be linked to the precursor ion of interest.Triple quadrupoles (3Qs) are the most universal instruments for quantitative LC/MS analyses and were employed in Studies I, II and III for this purpose. A 3Q consists of three sequential Qs (Q1q2Q3). Q1 and Q3 can be used as scanning or selecting analyzers while q2 is operated in the RF-only mode, as a wide-band pass filter for the ions or a collision cell when filled with a neutral collision gas such as nitrogen or argon. Several MS/MS modes, such as precursor ion, product ion or neutral loss scanning, and selected ion (SIM) or selected reaction monitoring (SRM), are available, depending on the Q1 and Q3 settings applied. In SRM Q1 and Q3 are static, providing enhanced selectivity and sensitivity which makes it the best choice for quantitative analyses, as used in Studies I, II and III. Product ion scanning (in which the ions of interest are selected by Q1, fragmented in q2 and fragments are scanned by Q3) was used in Study IV for the identification of FICZ metabolites.To enable the masses of fragment ions to be accurately measured, QqToF instruments have also been developed, in which the Q3 of a 3Q spectrometer is replaced with a ToF system. These instruments are routinely applied for accurate mass measurements of small molecules, e.g. metabolites of diverse compounds [112]. 4.2.2 Ion trapAn ion trap (IT) analyzer can be visualized as a Q wrapped around itself, composed of a ring electrode and two end-cap electrodes. The IT analysis procedure consists of ion injection, isolation, excitation and scanning. Ions with specific m/z values are selectively ejected by linearly increasing the amplitude of the RF applied to the ring electrodes [110]. An IT is a tandem-in-time system (while a 3Q system is tandem-in space), in which multiple stages of tandem mass spectrometry (MSn) can be performed. MSn experiments are very advantageous for the elucidation


of fragmentation pathways and were in used in Study I to examine the CID fragmentation behavior of OPEs. The trialkyl phosphorous esters were subjected to three successive neutral losses of their aliphatic chains until only protonated phosphoric acid was left. The CID fragmentation patterns of di- and tri-arylated phosphorous esters were however more complex [113]. To combine the MSn functionality with accurate mass and high resolution analysis, IT and ToF analyzers have been placed after one another in a single instrument [114].

4.2.3 Linear ion trapLinear ion trap (LIT) instruments combine the advantages of 3Q and IT instruments in the same platform, providing the benefits of both types of mass spectrometers in a single instrument. LIT has several advantages over conventional three-dimensional ITs, such as enhanced trapping efficiencies and higher ion storage capacities due to larger trapping volumes [115]. LITs have been successfully coupled to ToF [116] and FT-ICR [117] in LIT-ToF-MS and LIT-FT-ICR instruments, respectively, to combine ion accumulation and MSn features with the superior mass accuracy, resolution and sensitivity of ToF-MS or FT-ICR-MS.The LIT used in Study IV (Q TRAP, AB/MSD Sciex) is based on a 3Q platform in which Q3 can be operated in either the normal RF/DC mode or LIT mode [115]. The instrument provides all the scan modes that 3Qs offer as well as trap scan modes, such as: MS3; enhanced resolution (ER) mode, in which the slow scanning capability of the LIT component increases the resolution obtained; and enhanced product ion (EPI) mode [118]. In Study IV the EPI mode was employed to identify the monosulfated metabolites of FICZ in human urine samples due to the higher sensitivity it provides compared to 3Q instruments. The greater sensitivity afforded by LIT instruments originates from their higher duty cycles and ability to obtain product ion spectra below the low mass


cut-off, due to the injection solely of ions selected in Q1 into the trap and associated reductions in space charge problems [119]. These features have led to the use of LITs in many applications such as drug discovery [120] and proteomics [121].

4.2.4 Time of flight A linear time-of-flight (ToF) mass analyzer consists of an ion accelerating region and a flight tube. The charged analytes are accelerated in an electrical field before entering a field-free tube, in which the time to reach the detector is measured. At the start of the flight tube all the ions have been accelerated by the same potential difference and those with the same charge thus have gained the same kinetic energy from this potential. However, their velocities (and hence arrival times at the detector) will differ due to differences in their masses; an ion with a high mass will have a lower velocity than another ion with the same charge but lower mass. Thus, the time of flight of each ion depends on its m/z ratio. The major drawback of a linear ToF instrument is that it has insufficient mass resolution for many applications, due to small variations in the initial kinetic energies of ions of the same m/z, leading to differences in the flight times. To overcome this problem two techniques, “reflectance” using a reflectron and delayed extraction, are commonly employed and were applied in the MALDI-ToF instrument used in Studies II, III, V and VI. A reflectron is a series of rings or grids located after the flight tube used to focus ions of the same m/z but different kinetic energies. The reflectron creates a retarding field in which the ions enter to different depths depending upon their kinetic energies and are then reflected back to the flight tube. Ions of the same m/z but higher kinetic energies spend more time in the reflectron, whereas those with lower kinetic energy return to the flight tube more quickly, thus their spread is reduced and the resolution improved. In delayed extraction, ions drift without any electrical field for a certain time after formation,


and after this delay faster ions are accelerated less by the field gradient between the sample target and the extraction plate than the slower ones. This compensates for variations in the initial energy of ions with the same m/z and enhances resolution. The delayed extraction technique can be used in both linear and reflectron ToF instruments. In comparison to 3Q instruments, discussed above, ToF instruments provide higher sensitivity in full-scan mode since they generally have a higher transmission of ions than scanning analyzer. They also have a higher resolving power than 3Q instruments, which facilitates discrimination of analytes with close m/z ratios. It would have been very beneficial to use this feature in Study IV to measure the m/z ratios of the putative monosulfated-FICZ metabolites accurately, but no ToF instrument was accessible at the time.

4.3 DetectorsAfter ions have been produced in the ion source of a MS instrument and separated according to their m/z ratios in the analyzer, they must be detected. The most common kinds of detector in 3Q instruments are electron multiplier detectors [122] and photomultiplier tube detectors [123]. In a photomultiplier tube the ions exiting the mass analyzer are converted to electrons, which travel through an electron multiplier and each electron induces the emission of several secondary electrons.In ToF instruments the detector has to measure the differences in the flight times of the ions, thus a detector with faster responses is required. Micro channel plates (MCPs) [124], consisting of two-dimensional parallel arrays of miniaturized electron multipliers, are the detectors used today in ToF instruments.

47Conclusions and future perspectives

5. Conclusions and future perspectivesMass spectrometry is sometimes considered a generally applicable detection technique, but even with high-resolution instruments extensive sample clean-up, pre-concentration and separation of compounds prior to MS detection is often necessary. Careful attention must also be paid to the choice of ionization technique. In addition, the final extract obtained from sample pretreatment should be compatible with the techniques subsequently used to analyze the sample. In several of the studies this thesis based upon the hyphenated separation-detection technique LC/ MS was used (in various modes), for which rigorously cleaned-up samples are often required. SALDI-MS was also applied, and clean samples were also essential in this context, since no prior separation of analytes was performed and the presence of unwanted compounds would have greatly hindered their detection. Thus, a number of sample preparation procedures were developed, using selective materials for SPE. A combined size exclusion-reversed phase material, RAM, was used for the successful on-line extraction of OPEs from human plasma in Study I. The cost of RAM pre-columns is relatively high, but their ease of use and the possibility they provide for repeated on-line use made them a good choice for cleaning up complex matrices such as plasma. However, despite obtaining relatively clean extracts, use of an internal standard was still essential when quantifying the analytes with LC/MS. The internal standard used here was adequate, but more accurate quantification might have been attained if an isotope of one of the analytes of interest had been used, or (ideally) an isotopically-labeled internal standard had been used for each analyte. The extent of matrix effects in two different ionization techniques, ESI and APCI, were compared and APCI was found to be the better option.Another selective material used for SPE was GCB, in µ-trap and disk formats in Studies II and III, respectively. GCB functioned very well as a mixed mode sorbent for the extraction of a wide range of compounds,

48 Conclusions and future perspectives

including pesticides, human medicines, sulfonamides and OPEs in water and urine. A new property of GCB was also discovered and applied in Study II; its utility as a SALDI surface. This led to the development of a novel screening procedure, in which both its SPE and SALDI properties were exploited, to eliminate negative samples prior to further analyses and quantification. Using GCB in the disk format in Study III, the entire SPE-SALDI-MS can be performed automatically and is the subject of further investigation. Application for a patent regarding the GCB disk and automated screening-analyses is currently in progress. In addition, it is possible to perform a more informative screening if an LDI-MS/MS instrument is available. In Study IV, in which no selective extraction was applied, the LC separation efficiency was improved to overcome problems associated with determining analytes in complex matrices (more specifically FICZ metabolites in human urine). The separation might have been further enhanced by using a UHPLC or MDLC system. However, in this case a column with smaller than conventional (1.8 µm) particles was used at high temperature. It was not possible to identify a number of sulfated-FICZ metabolites due to lack of standards, as well as instrumentation limitations. By accurately measuring the m/z of these ions and their fragments with a high mass resolution instrument, such as a QqToF, it would be possible to identify the relevant metabolites to some extent. However, it would still not be possible to unambiguously identify them by MS without appropriate standards. It would also be interesting to quantify the metabolites of interest, which could be done by means of a nitrogen chemiluminescence detector, even in the absence of the relevant standards.Apart from GCB, two new SALDI surfaces suitable for analyses of small molecules were developed. The first surface, oxidized GCB (Paper V), was obtained by increasing the number of cationization sites on GCB (by oxidation) which enhanced the positive ionization mode

49Conclusions and future perspectives

SALDI-MS signal-to-noise ratios of small molecules. The second was a hard ceramic surface, silicon nitride (Paper VI). The addition of functional groups to SALDI surfaces is an interesting option that requires further research. For instance, attempts to improve surfaces for negative mode SALDI-MS surfaces can be made by coupling electron donating functional groups to surfaces of suitable substances. Another feature of oxidized GCB nanoparticles, their ability to form a stable suspension in water, may be beneficial in SALDI-MS imaging, since it would be possible to electrospray a homogeneous layer of oxidized GCB on the subject to be imaged, e.g. a slice of body tissue.

50 Acknowledgments

6. AcknowledgmentsI have met many fantastic people during this long education journey, which started in Iran, continued in Australia and ended in Sweden! Every one of them has contributed to this work in a way and I am grateful to all.I would like to thank the two supervisors I have had during my PhD studies, Carlo Crescenzi and Gunnar Thorsén, for all the support, ideas and advice they have given me. I would also like to acknowledge the people I have collaborated with. Ulf and Agneta Rannug, thanks for introducing me to, and involving me in, your extensive research on ‘FICZ’. Emma Wincent, you have answered all my ‘biology’ questions with a lot of patience and been a lovely friend for me, thank you. Leopold Ilag, thanks for all the fruitful discussions, and Shahram, we have shared a lot of happy (articles being accepted) and frustrating (adding standards to extracted solutions after a very long day of extracting samples!!!) moments, I hope we continue to collaborate.I am grateful to all the people I have met during the time I have spent in the Department of Analytical Chemistry: Anders, Ulrika (thanks for openly sharing your knowledge of mass spectrometry), Anita, Anne-Marie, Jonas (thanks for all the nice chats as well as solving my computer-related problems), Ulla, Roger, Conny, Lena, Bosse, Björn, Ninni, Sven, Ralf, Magnus, Theres, Håkan, Ramon, Cristina (it was unfortunate that you did not stay in Sweden), Ove (thanks for always being so helpful), Anders Ch., Jonas B., Magnus E., Yvonne, Kristina C., Malin (thanks for all your encouragement), Jenny, Johanna (thanks for all the advice on raising children), Petter T., Sindra, Helena I. (thanks for all the help with the triple quadrupole instruments), Kristina M. (you are the first person I met in the department and I will never forget all your kindness), Helena H. (thanks for always listening and giving good advice), Caroline (thanks for being such a fantastic room mate), Leila (thanks for your friendship), Axel, Gianluca, Thuy, Christoffer, Silvia,

51Acknowledgments

Zdenek, Yasar, Erik, Fredrik, Leo, Tuula, Petter O., Liying, Aljona and Ragnar. I would also like to express my appreciation to my parents. You have always encouraged and believed in me. You have supported and helped me in every possible way, especially during the writing of this thesis. A very big thank you. Negar and Pouya, you have always been there for me even if we live in three different countries! Talking to you two always gives me the strength to go on. Arvid and Alvin, you two show me the true meaning of life and your contributions to this thesis are beyond words. Steven, none of this would have ever been possible without you and your devotion to our family. Tack så mycket alla!

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