Accurate mass-based analysis in human sport doping control : Application of liquid chromatography - time-of-flight mass spectrometry

Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.

Photosynthesis of ground vegetation in boreal Scots pine forests

Research on carbon uptake in boreal forests has mainly focused on mature trees, even though ground vegetation species are effective assimilators and can substantially contribute to the CO2 uptake of forests. Here, I examine the photosynthesis of the most common species of ground vegetation in a series of differently aged Scots pine stands, and at two clear-cut sites with substantial differences in fertility. In general, the biomass of evergreen species was highest at poor sites and below canopies, whereas grasses and herbs predominated at fertile sites and open areas. Unlike mosses, the measured vascular species showed clear annual cycles in their photosynthetic activity, which increased earlier and decreased later in evergreen vascular species than in deciduous species. However, intraspecific variation and self-shading create differences in the overall level of photosynthesis. Light, temperature history, soil moisture and recent possible frosts could explain the changes in photosynthesis of low shrubs and partially also some changes in deciduous species. Light and the occurrence of rain events explained most of the variation in the photosynthesis of mosses. The photosynthetic production of ground vegetation was first upscaled, using species-specific and mass-based photosynthetic activities and average biomass of the site, and then integrated over the growing season, using changes in environmental factors. Leaf mass-based photosynthesis was highest in deciduous species, resulting in notably higher photosynthetic production at fertile sites than at poor clear-cut sites. The photosynthetic production decreased with stand age, because flora changed towards evergreen species, and light levels diminished below the canopy. In addition, the leaf mass-based photosynthetic activity of some low shrubs declined with the age of the surrounding trees. Different measuring methods led to different momentary rate of photosynthesis. Therefore, the choice of measuring method needs special attention.

End-User Attitudes Towards EDM Use in Construction Project Work: Case Study

This article reports on a cross-sectional case study of a large construction project in which Electronic document management (EDM) was used. Attitudes towards EDM from the perspective of individual end users were investigated. Responses from a survey were combined with data from system usage log files to obtain an overview of attitudes prevalent in different user segments of the total population of 334 users. The survey was followed by semi-structured interviews with representative users. A strong majority of users from all segments of the project group considered EDM as a valuable aid in their work processes, despite certain functional limitations of the system used and the complexity of the information mass. Based on the study a model describing the key factors affecting end user EDM adoption is proposed. The model draws on insight from earlier studies of EDM enabled projects and theoretical frameworks on technology acceptance and success of information systems, as well as the insights gained from the case study.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Nitrogen dynamics of organic farming in a crop rotation based on red clover (Trifolium pratense) leys

In agricultural systems which rely on organic sources of nitrogen (N), of which the primary source is biological N fixation (BNF), it is extremely important to use N as efficiently as possible with minimal losses to the environment. The amount of N through BNF should be maximised and the availability of the residual N after legumes should be synchronised to the subsequent plant needs in the crop rotation. Six field experiments in three locations in Finland were conducted in 1994-2006 to determine the productivity and amount of BNF in red clover-grass leys of different ages. The residual effects of the leys for subsequent cereals as well as the N leaching risk were studied by field measurements and by simulation using the CoupModel. N use efficiency (NUE) and N balances were also calculated. The yields of red clover-grass leys were highest in the two-year-old leys (6 700 kg ha-1) under study, but the differences between 2- and 3-year old leys were not high in most cases. BNF (90 kg ha-1 in harvested biomass) correlated strongly with red clover dry matter yield, as the proportion of red clover N derived from the atmosphere (> 85%) was high in our conditions of organically farmed field with low soil mineral N. A red clover content of over 40% in dry matter is targeted to avoid negative N-balances and to gain N for the subsequent crop. Surprisingly, the leys had no significant effect on the yields and N uptake of the two subsequent cereals (winter rye or spring wheat, followed by spring oats). On the other hand, yield and C:N of leys, as well as BNF-N and total-N incorporated into the soil influenced on subsequent cereal yields. NUE of cereals from incorporated ley crop residues was rather high, varying from 30% to 80% (mean 48%). The mineral N content of soil in the profile of 0-90 cm was low, mainly 15-30 kg ha-1. Simulation of N dynamics by CoupModel functioned satisfactorily and is considered a useful tool to estimate N flows in cropping systems relying on organic N sources. Understanding the long-term influence of cultivation history and soil properties on N dynamics remains to be a challenge to further research.

Polymerization of Ethene and Branched α-olefins with Group IV Metal Complexes Bearing Nitrogen- and Oxygen-Based Ligands

The commodity plastics that are used in our everyday lives are based on polyolefin resins and they find wide variety of applications in several areas. Most of the production is carried out in catalyzed low pressure processes. As a consequence polymerization of ethene and α-olefins has been one of the focus areas for catalyst research both in industry and academia. Enormous amount of effort have been dedicated to fine tune the processes and to obtain better control of the polymerization and to produce tailored polymer structures The literature review of the thesis concentrates on the use of Group IV metal complexes as catalysts for polymerization of ethene and branched α-olefins. More precisely the review is focused on the use of complexes bearing [O,O] and [O,N] type ligands which have gained considerable interest. Effects of the ligand framework as well as mechanical and fluxional behaviour of the complexes are discussed. The experimental part consists mainly of development of new Group IV metal complexes bearing [O,O] and [O,N] ligands and their use as catalysts precursors in ethene polymerization. Part of the experimental work deals with usage of high-throughput techniques in tailoring properties of new polymer materials which are synthesized using Group IV complexes as catalysts. It is known that the by changing the steric and electronic properties of the ligand framework it is possible to fine tune the catalyst and to gain control over the polymerization reaction. This is why in this thesis the complex structures were designed so that the ligand frameworks could be fairly easily modified. All together 14 complexes were synthesised and used as catalysts in ethene polymerizations. It was found that the ligand framework did have an impact within the studied catalyst families. The activities of the catalysts were affected by the changes in complex structure and also effects on the produced polymers were observed: molecular weights and molecular weight distributions were depended on the used catalyst structure. Some catalysts also produced bi- or multi-modal polymers. During last decade high-throughput techniques developed in pharmaceutical industries have been adopted into polyolefin research in order to speed-up and optimize the catalyst candidates. These methods can now be regarded as established method suitable for both academia and industry alike. These high-throughput techniques were used in tailoring poly(4-methyl-1-pentene) polymers which were synthesized using Group IV metal complexes as catalysts. This work done in this thesis represents the first successful example where the high-throughput synthesis techniques are combined with high-throughput mechanical testing techniques to speed-up the discovery process for new polymer materials.

European aspen and hybrid aspen under changing environment : Leaf traits, growth and litter decomposition

Understanding the responses of species and ecosystems to human-induced global environmental change has become a high research priority. The main aim of this thesis was to investigate how certain environmental factors that relate to global change affect European aspen (Populus tremula), a keystone species in boreal forests, and hybrid aspen (P. tremula × P. tremuloides), cultivated in commercial plantations. The main points under consideration were the acclimatization potential of aspen through changes in leaf morphology, as well as effects on growth, leaf litter chemistry and decomposition. The thesis is based on two experiments, in which young aspen (< 1 year) were exposed either to an atmospheric pollutant [elevated ozone (O3)] or variable resource availability [water, nitrogen (N)]; and two field studies, in which mature trees (> 8 years) were growing in environments exposed to multiple environmental stress factors (roadside and urban environments). The field studies included litter decomposition experiments. The results show that young aspen, especially the native European aspen, was sensitive to O3 in terms of visible leaf injuries. Elevated O3 resulted in reduced biomass allocation to roots and accelerated leaf senescence, suggesting negative effects on growth in the long term. Water and N availability modified the frost hardening of young aspen: High N supply, especially when combined with drought, postponed the development of frost hardiness, which in turn may predispose trees to early autumn frosts. This effect was more pronounced in European aspen. The field studies showed that mature aspen acclimatized to roadside and urban environments by producing more xeromorphic leaves. Leaf morphology was also observed to vary in response to interannual climatic variation, which further indicates the ability of aspen for phenotypic plasticity. Intraspecific variation was found in several of the traits measured, although intraspecific differences in response to the abiotic factors examined were generally small throughout the studies. However, some differences between clones were found in sensitivity to O3 and the roadside environment. Aspen leaf litter decomposition was retarded in the roadside environment, but only initially. By contrast, decomposition was found to be faster in the urban than the rural environment throughout the study. The higher quality of urban litter (higher in N, lower in lignin and phenolics), as well as higher temperature, N deposition and humus pH at the urban site were factors likely to promote decay. The phenotypic plasticity combined with intraspecific variation found in the studies imply that aspen has potential for withstanding environmental changes, although some global change factors, such as rising O3 levels, may adversely affect its performance. The results also suggest that the multiple environmental changes taking place in urban areas which correspond closely with the main drivers of global change can modify ecosystem functioning by promoting litter decomposition, mediated partly by alterations in leaf litter quality.

Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation

This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of microchannel, which leads to a sharp electrospray tip. Microchannel contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on-chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Heated Nebulizer Microchips for Mass Spectrometry

Miniaturized analytical devices, such as heated nebulizer (HN) microchips studied in this work, are of increasing interest owing to benefits like faster operation, better performance, and lower cost relative to conventional systems. HN microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically a few µL/min, and have previously been utilized as ion sources for mass spectrometry (MS). Conventional ion sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. First, a new method for thermal and fluidic characterization of the HN microchips was developed and used to study the chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip atmospheric pressure photoionization (APPI) high resolution mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the MS instrument. Thanks to its suitability to high temperature, microchip APPI provided efficient vaporization of nonvolatile compounds in crude oil. The first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved by applying only high (sonic) speed nebulizer gas to an HN microchip. SSI significantly broadens the range of analytes ionizable with the HN chips, from small stable molecules to labile biomolecules. The analytical performance of the microchip SSI source was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds whereas with the most popular electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented analytical methods showed good performance. The first integrated LC–HN microchip was developed and presented. In a single microdevice, there were structures for a packed LC column and a heated nebulizer. Nonpolar and polar analytes were efficiently ionized by APPI. Ionization of nonpolar and polar analytes is not possible with previously presented chips for LC–MS since they rely on ESI. Preliminary quantitative performance of the new chip was evaluated and the chip was also demonstrated with optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented. The DAPPI technique is based on an HN microchip providing desorption of analytes from a surface. Photons from a photoionization lamp ionize the analytes via gas-phase chemical reactions, and the ions are directed into an MS. Rapid analysis of pharmaceuticals from tablets was successfully demonstrated as an application of DAPPI.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.