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Analysis of biological samples can be a difficult task. This thesis covers a broad aspect of the analytical areas of capillary electrophoresis (CE) and capillary electrochromatography (CEC) in combination with mass spectrometry (MS) that are of great importance for achieving fast, accurate and sensitive bioanalyses. A significantly time reduced and automated system for sample cleanup was developed to greatly simplify the pretreatment process of biological samples with a complex matrix. Desalting and preconcentration of species in urine was conducted and the limit of detection for the antidepressant escitalopram was lowered 10 times. This extraction devise was also successfully incorporated in a chip based platform for the possibility to be a part of multidimensional separation systems. The reduced risk of sample loss leads to improved detection limits, which are usually one the most challenging parts when working with bioanalyses. In the area of separation, a monomer surface with tailored hydrophobicity was developed to achieve rapid, high efficient separations of complex mixtures. Within five minutes a tryptic digest of a protein could be separated and then identified by a Mascot search. The applications addressed have been focused on medical conditions which are of highest interest for both physicians and patients. A high throughput analysis of the kynurenine metabolites with CE-MS offers a new method to rapidly examine samples from patients with neurological disorders. A screening study of possible biomarkers for the two different types of appendicitis, gangraenous and phlegmonous was conducted. Indicative patterns were found for both pre and post surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis. A preliminary study of polycystic ovary syndrome also offered some valuable results for future biomarker identification.

Pour aider à la protection de l’environnement et au diagnostic de maladies, il est nécessaire de disposer de méthodes d’analyse performantes. Ces méthodes doivent être rapides et peu coûteuses afin d’analyser un grand nombre d’échantillons et détecter la présence éventuelle de la molécule recherchée. C’est dans ce cadre qu’un bio-essai à double reconnaissance dédié aux petites molécules a été développé. Il repose sur l’utilisation d’aptamères comme élément de reconnaissance moléculaire et sur la formation de « kissing complex ». Les aptamères sont des oligonucléotides qui possèdent une grande spécificité et une grande affinité pour leur cible. Les « kissing complexes » désignent, quant à eux, l’interaction de deux séquences au niveau de leur boucle apicale. L’objectif de la thèse est de continuer l’étude et l’optimisation de ce schéma d’analyse, puis montrer sa potentialité et sa versatilité. Dans un 1er temps, la détection en multiplexe de plusieurs petites molécules a été mise en place. L’optimisation du système de détection a ensuite été effectuée pour la détection de l’adénosine avec le développement d’une nouvelle stratégie de détection. Enfin, un test colorimétrique basé sur l’utilisation de nanoparticules d’or a été décrit. / Bioanalytical tools based on molecular recognition elements constitute one of the most important strategies used to routinely quantify low molecular weight analytes in biological fluids or environmental matrices as disease-related biomarkers, drugs or toxins/contaminants. Recently, a novel aptamer-based sensing design has been reported. It is based on a dual recognition mechanism that involves the formation of functional nucleic acid architectures, named “kissing complex”. Aptamers are single stranded oligonucleotides that possess high affinity and high selectivity for their target. The aim of this thesis is to pursue the study and the optimization of this sandwich like scheme and to prove its potential. Firstly, the simultaneous detection of multiple small analytes by fluorescence anisotropy using the sandwich like scheme has been reported. Then, this novel bioassay has been optimized through the development of a new strategy. Finally, a gold nanoparticle colorimetric

<div><div><div><p>To prolong their long-term stability, protein molecules are commonly dispensed as lyophilized powders to be reconstituted before use. Evaluating the stability of these biomolecules in the solid state is routinely done by using various analytical techniques such as glass transition temperature, residual moisture content and other spectroscopic techniques. However, these techniques often show poor correlation with long term storage stability studies. As a result, time intensive long term storage stability studies are still the golden standard for evaluating protein formulations in the solid state. Over the past few years, our lab has developed solid-state hydrogen deuterium exchange- mass spectrometry (ssHDX-MS) as an analytical tool that probes the backbone of a protein molecule in the solid state. ssHDX-MS gives a snapshot of protein-matrix interactions in the solid state and has a quick turnaround of a few weeks as opposed to a few months for accelerated stability testing. Additionally, various studies in the past have demonstrated that ssHDX-MS can be used for a wide range of biomolecules and shows strong correlation to long term stability studies routinely employed.</p><p>The main aim of this dissertation is to provide an initial understanding of the mechanism behind ssHDX-MS in structured protein formulations. Specifically, this dissertation is an attempt at studying the effects of various experimental variables on the ssHDX-MS of myoglobin formulations as well as demonstrating the utility of this analytical technique. Firstly, the effects of varying temperature and relative humidity on ssHDX-MS of myoglobin formulations is studied with the help of statistical modeling. Secondly, the effects of pressure on ssHDX-MS of myoglobin formulations are evaluated at an intact and peptide digest levels. Finally, ssHDX-MS is used as a characterization tool to evaluate the effects of two different lyophilization methods on the structure and stability of myoglobin formulations. The results of studies described in this dissertation show ssHDX-MS to be sensitive to changes in experimental parameters, namely temperature, relative humidity, pressure, and excipients. Additionally, ssHDX-MS results were in good agreement with other routinely employed analytical and stability testing techniques when used to compare the effects of two lyophilization methods on myoglobin formulations.</p></div></div></div>

Bioanalytical chemistry is a challenging field, often involving complex samples, such as blood, plasma, serum or urine. In many applications, sample cleanup is the most demanding and time-consuming step. In the work underlying this thesis a novel dynamic miniature extractor, known as a hollow-fibre liquid-phase microextractor (HF-LPME), was designed, evaluated and studied closely when used to clean plasma samples. Aqueous-organic-aqueous liquid extraction, in which the organic liquid is immobilised in a porous polypropylene membrane, was the principle upon which the extractor was based, and this is discussed in all the papers associated with this thesis. This type of extraction is known as supported-liquid membrane extraction (SLM). The aim of this work was the development of a dynamic system for SLM. It was essential that the system could handle small sample volumes and had the potential for hyphenations and on-line connections to, for instance, LC/electrospray-MS. The design of a miniaturised HF-LPME device is presented in Paper I. The extraction method was developed for some weakly acidic pesticides and these were also used for evaluation. In the work described in Paper II, the method was optimised on the basis of an experimental design using spiked human plasma samples. Paper III presents a detailed study of the mass-transfer over the liquid membrane. The diffusion through the membrane pores was illustrated by a computer-simulation. Not surprisingly, the more lipophilic, the greater the retention of the compounds, as a result of dispersive forces. The main focus of the work described in Paper IV was to make the HF/LPME system more versatile and user-friendly; therefore, the extractor was automated by hyphenation to a SIA system. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.

New solid phase microextraction (SPME) method was developed for brain tissue bioanalysis on a liquid chromatography mass spectrometry platform. To achieve set objectives, in vivo SPME desorption process was optimized for high throughput analysis through the development of a desorption device. Subsequently, new SPME coatings were developed for the extraction of polar neurotransmitters from biological matrices. In a targeted analysis, in vivo SPME was used to monitor of changes in the concentrations of endogenous compounds (multiple neurotransmitters) and exogenous drugs (carbamazepine and cimetidine) in the striatum of the rat brain extracellular fluid. For the first time, SPME was used for quantitative analysis of neurotransmitters and also study spacial distribution of other drugs in different regions of the brain extracellular fluid. A new approach was developed for improved metabolites coverage in a global non-targeted metabolomics studies. The proposed in vivo method showed how complementary results can be obtained through the combination of microdialysis and SPME for simultaneous sampling of the brain extracellular fluid. Finally, in a clinical application, SPME was used to monitor changes in the concentration of multiple neurotransmitters during deep brain stimulation of the pre-frontal cortex of the brain.

The goal of this research work was to understand the clinical-pharmacology based treatment approaches for sorafenib. Treatment with sorafenib is associated with high inter-patient variability in pharmacokinetic exposures, efficacy and toxicity. We explored the demographic, laboratory, clinical and pharmacogenetic factors to elucidate the sources of variability. In addition, we examined the impact of pharmacogenetic variation in VEGFR2, an important mediator of the VEGF pathway, on risk of prostate cancer. To support these investigations, (mainly single-dose) pharmacokinetic, pharmacogenetic, efficacy and toxicity information were collected from patients with solid tumors, enrolled in five phase I / II clinical trials at National Cancer Institute. Non-compartmental analysis-general linear modeling (NCA-GLM), population pharmacokinetic analysis and several correlative studies were performed to characterize the sources of variability in pharmacokinetics and response. The role of prostate specific antigen (PSA) and ex-vivo anti-angiogenic activity as efficacy markers was evaluated, respectively, for patients with prostate cancer treated with sorafenib and patients with solid tumors treated with combination of sorafenib and bevacizumab. Sweat concentrations of sorafenib were measured to study its association with development of hand-foot skin reaction (HFSR). Only body weight was a significant covariate for volume of distribution by population pharmacokinetic analysis, while BSA, albumin and UGT1A9*3 appeared to be significant by NCA-GLM. However, the contribution of these covariates in overall exposure variability was very small; hence, these were considered clinically irrelevant. The association of sorafenib exposure with efficacy in patients with prostate cancer, colorectal cancer and combined solid tumors were not significant; exposure-efficacy relationship for lung cancer patients requires further evaluation. Sorafenib exposures appeared to be associated with incidences of rash in single agent trials and with HFSR in trials involving treatment with sorafenib and bevacizumab combination. In-vitro cell-line experiments determined that prostate specific antigen (PSA) is not a suitable marker of efficacy in patients with prostate cancer treated with sorafenib. The ex-vivo anti-angiogenic activity, measured by rat-aortic ring assay using patient serum samples, appeared to be not associated with clinical response. Sorafenib concentration in sweat, upto ≥5 ng/mL, apparently was not associated with HFSR. The VEGFR2 H472Q polymorphism was associated with progression-free survival (PFS) (with an apparent heterozygous advantage for survival) and toxicities in patients treated with drugs against the VEGF pathway. Patients who developed hypertension and HFSR on bevacizumab and sorafenib therapy, respectively, appeared to have longer PFS. Therefore, these side effects should be effectively managed to avoid/delay the treatment discontinuation. The VEGFR2 H472Q and V297I genotype were not predictive of risk of prostate cancer in Caucasian subjects.

Measuring urinary tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide conjugate may provide the best biomarker of tobacco smoke lung carcinogen metabolism. Existence of differences in the extent of NNAL metabolism rates may be potentially related to an individuals’ lung cancer susceptibility. Low concentrations of NNAL in smokers urine (<1 ng/mL) require sensitive and selective methods for analysis. Traditionally, this involves extensive, time-consuming sample preparation that limits throughput and adds to measurement variability. Molecularly imprinted polymers (MIPs) have been developed for the analysis of urinary NNAL by offline cartridge extraction combined with LC-MS/MS. This method when reproduced demonstrated problems with matrix effects. In the first part of this work, investigation of matrix effects and related problems with sensitivity for the published offline extraction method has been conducted. In order to address the need to improve throughput and other analytical figures of merit for the original method, the second part of this work deals with development of a high-throughput online microfluidic method using capillary-columns packed with MIP beads for the analysis of urinary NNAL. The method was validated as per the FDA guidance, and enabled low volume, rapid analysis of urinary NNAL by direct injection on a microfluidic column packed with NNAL specific MIP beads. The method was used for analysis of urinary NNAL and NNAL-Gluc in smokers. Chemometric methods were used with this data to develop a potential cancer-risk-assessment tool based on pattern recognition in the concentrations of these compounds in urine. In the last part, method comparison approaches for the online and the offline sample extraction techniques were investigated. A ‘fixed’ range acceptance criterion based on combined considerations of method precision and accuracy, and the FDA bioanalytical guidance limits on precision and accuracy was proposed. Data simulations studies to evaluate the probabilities of successful transfers using the proposed criteria were performed. Various experimental designs were evaluated and a design comprised of 3 runs with 3 replicates each with an acceptance range of ±20% was found appropriate. The off-line and the on-line sample extraction methods for NNAL analysis were found comparable using the proposed fixed range acceptance criteria.

The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices. Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices. Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary. In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies. The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma.

Abstract Drug metabolism is one of the most important events a drug faces after administration. Traditionally, drug metabolism has only been considered as a major clearance and elimination step in the pharmacokinetics of a drug. However, drug metabolism is also one of the important factors behind safety and toxicity issues in drug discovery and development. Some of the mechanisms behind metabolism-related toxicity we do understand well while others, especially the role of reactive metabolites, need further research. The thesis reviews the role of drug metabolism in the drug discovery and development process from the point of view of metabolism and metabolites. Special emphasis is put on reviewing the metabolism behind human toxicity and safety, and the roles of circulating and reactive metabolites in particular. Ospemifene is a nonsteroidal selective estrogen receptor modulator recently approved for the treatment of vulvar and vaginal atrophy in postmenopausal women with moderate to severe dyspareunia. The present study characterized the in vitro and in vivo metabolism and potential drug interactions of ospemifene. The principal human metabolites were identified and the adequacy nonclinical animal exposure was evaluated. The major human cytochrome P450 enzymes involved in the formation of principal metabolites were also identified and the clinical consequences assessed. Finally, the interaction potential of ospemifene as a cytochrome P450 enzyme inducer or inhibitor was investigated. As a result, ospemifene was considered to be safe drug from a metabolic interaction point of view. This study was part of the drug development program of ospemifene and practically all of the in vitro study data were included in the marketing authorization application of ospemifene. Ospemifene was also a case molecule in the development of new methodologies to study drug metabolism and drug-drug interactions. / Tiivistelmä Lääkeainemetabolia on lääkeaineen farmakokinetiikassa tärkeä puhdistuma- ja eliminaatioaskel, jonka rooli on ymmärretty varsin hyvin. Lääkeainemetabolialla on myös merkittävä vaikutus lääkeaineen toksisuuteen ja lääkkeen käytön turvallisuuteen. Osa lääkeainemetaboliaan liittyvistä toksisuusmekanismeista selvitetty hyvin, mutta erityisesti reaktiivisiin metaboliitteihin liittyvä osa vaatii vielä tutkimusta. Tämän työn kirjallisuusosassa katselmoidaan lääkeainemetabolian merkitystä lääkekehitysprosessissa painottaen erityisesti lääkeainemetabolian sekä reaktiivisten ja verenkierrossa kiertävien metaboliatuoteiden vaikutusta toksisuuteen ihmisellä ja merkitystä turvalliseen lääkkeiden käyttöön. Ospemifeeni on uusi ei-steroidinen selektiivinen estrogeenireseptorimodulaattori, joka on hyväksytty yhdynnänaikaisesta kivusta kärsivien postmenopausaalisten naisten vulvan ja vaginan limakalvojen kuivumisen hoitoon. Tässä tutkimuksessa selvitettiin ospemifeenin lääkeainemetaboliaa ihmisellä ja koe-eläimillä sekä mahdollisia lääkeinteraktioita. Tutkimuksessa tunnistettiin tärkeimmät metaboliitit ihmisellä ja arvioitiin eläinkokeissa käytettyjen koe-eläinten altistumisen kattavuus niille. Työssä selvitettiin myös tärkeimmät päämetaboliitteja katalysoivat sytokromi P450 -entsyymit ja arvioitiin löydösten kliinistä merkitystä. Lisäksi tutkittiin aiheuttaako ospemifeeni lääkeinteraktioita muille lääkeaineille indusoimalla tai inhiboimalla sytokromi P450 -entsyymejä. Tutkimustulosten perusteella ospemifeenia voidaan pitää lääkeainemetabolian suhteen turvallisena lääkkeenä. Tämä tutkimus oli osa ospemifeenin lääkekehitysohjelmaa ja käytännössä kaikki tutkimustyön in vitro -tietoaineisto oli mukana ospemifeenin myyntilupa-hakemuksissa lääketurvallisuusviranomaisille. Ospemifeenia käytettiin tutkimustyön aikana myös yhtenä esimerkkimolekyylinä kehitettäessä uusia menetelmiä lääkeainemetabolian ja lääkeinteraktioiden tutkimiseen.