<p>The present thesis proposes new methods for obtaining objective and accurate diagnoses in modern healthcare. Non-invasive techniques have been used to examine or diagnose three different medical conditions, namely neuropathy among diabetics, radiotherapy induced erythema (skin redness) among breast cancer patients and diagnoses of cutaneous malignant melanoma. The techniques used were Near-InfraRed spectroscopy (NIR), Multi Frequency Bio Impedance Analysis of whole body (MFBIA-body), Laser Doppler Imaging (LDI) and Digital Colour Photography (DCP).</p><p>The neuropathy for diabetics was studied in papers I and II. The first study was performed on diabetics and control subjects of both genders. A separation was seen between males and females and therefore the data had to be divided in order to obtain good models. NIR spectroscopy was shown to be a viable technique for measuring neuropathy once the division according to gender was made. The second study on diabetics, where MFBIA-body was added to the analysis, was performed on males exclusively. Principal component analysis showed that healthy reference subjects tend to separate from diabetics. Also, diabetics with severe neuropathy separate from persons less affected.</p><p>The preliminary study presented in paper III was performed on breast cancer patients in order to investigate if NIR, LDI and DCP were able to detect radiotherapy induced erythema. The promising results in the preliminary study motivated a new and larger study. This study, presented in papers IV and V, intended to investigate the measurement techniques further but also to examine the effect that two different skin lotions, Essex and Aloe vera have on the development of erythema. The Wilcoxon signed rank sum test showed that DCP and NIR could detect erythema, which is developed during one week of radiation treatment. LDI was able to detect erythema developed during two weeks of treatment. None of the techniques could detect any differences between the two lotions regarding the development of erythema.</p><p>The use of NIR to diagnose cutaneous malignant melanoma is presented as unpublished results in this thesis. This study gave promising but inconclusive results. NIR could be of interest for future development of instrumentation for diagnosis of skin cancer.</p>

<p>The thesis describes the synthesis of chromatographic media using several different approaches, their characterizations and applications in liquid chromatography. The steps to achieve a separation column for a specific analyte are presented. The main focus of the study was the design of novel molecularly imprinted polymers.</p><p>Attachment of monolithic polymeric substrates to the walls of fused silica capillaries was studied in Paper I. With a broad literature survey, a set of common methods were tested by four techniques and ranked by their ability to improve anchoring of polymers. The best procedure was thus used for all further studies.</p><p>Synthesis of monoliths in capillary columns was studied in Paper II. With the goal of separating proteins without denaturation, various monoliths were polymerized in situ using a set of common monomers and cross-linkers mixed with poly(ethylene glycol) as porogen. The resulting network was expected to present “protein-friendly pores”. Chemometrics were used to find and describe a set of co-porogens added to the polymerization cocktails in order to get good porosity and flow-through properties.</p><p>Assessment of the macroporous structure of a monolith was described in Paper III. An alternative method to mercury intrusion porosimetry was proposed. The capillaries were embedded in a stained resin and observed under transmission electron microscope. Images were then computed to determine the pore sizes.</p><p>Synthesis of molecularly imprinted polymers grafted to a core mono-lith in a capillary was described in Paper IV. The resulting material, imprinted with local anaesthetics, was tested for its chromatographic performance. Similar imprinted polymers were characterized by microcalorimetry in Paper V. Finally, imprinted monoliths were also synthesized in a glass tube and further introduced in a NMR rotor to describe the interactions between stationary phase and template in Paper VI.</p>

<p>This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions.</p><p>The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations.</p><p>The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads.</p><p>In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.</p>

<p>A trend in natural science and especially in analytical chemistry is the increasing need for analysis of a large number of complex samples with low analyte concentrations. Biological samples (urine, blood, plasma, cerebral spinal fluid, tissue etc.) are often suitable for analysis with liquid separation mass spectrometry (LS-MS), resulting in two-way data tables (time vs. m/z). Such biological 'fingerprints' taken for all samples in a study correspond to a large amount of data. Detailed characterization requires a high sampling rate in combination with high mass resolution and wide mass range, which presents a challenge in data handling and exploration. This thesis describes methods for managing and exploring large data sets made up of such detailed 'fingerprints' (represented as data matrices). </p><p>The methods were implemented as scripts and functions in Matlab, a wide-spread environment for matrix manipulations. A single-file structure to hold the imported data facilitated both easy access and fast manipulation. Routines for baseline removal and noise reduction were intended to reduce the amount of data without loosing relevant information. A tool for visualizing and exploring single runs was also included. When comparing two or more 'fingerprints' they usually have to be aligned due to unintended shifts in analyte positions in time and m/z. A PCA-like multivariate method proved to be less sensitive to such shifts, and an ANOVA implementation made it easier to find systematic differences within the data sets.</p><p>The above strategies and methods were applied to complex samples such as plasma, protein digests, and urine. The field of application included urine profiling (paracetamole intake; beverage effects), peptide mapping (different digestion protocols) and search for potential biomarkers (appendicitis diagnosis) . The influence of the experimental factors was visualized by PCA score plots as well as clustering diagrams (dendrograms).</p>

This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions. The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations. The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads. In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.

This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy. Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed. Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (&lt;1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (&lt;1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.

Vatten är ett livsmedel som vi kommer i kontakt med dagligen. För att inte råka ut för sjuk-domar och infektioner renas dricksvattnet på vattenverken, främst för att reducera antalet patogener, d.v.s. sjukdomsframkallande mikroorganismer. Man brukar prata om tre grupper mikroorganismer i vattenrening; bakterier, virus och parasiter. Dessa grupper är vitt skilda i många avseenden och reduceras därför olika bra av olika reningssteg. Ju mer kunskap man kan få om reningsprocessen, desto bättre kan reningen optimeras. Därmed minimeras riskerna för konsumenterna att drabbas av infektioner. I det här arbetet används två modeller för att beskriva och utvärdera Borgs vattenverk som drivs av Norrköping Vatten AB. Modellen God desinfeksjonspraksis (GDP) är ett resultat av ett norskt projekt och baserar sig på formler och tabeller. Först avgörs råvattenkvaliteten genom att vattenverkets storlek och förekomst av mikroorganismer bedöms. Därefter görs diverse avdrag för förebyggande åtgärder, rening utöver desinfektion m.m. Slutligen bedöms desinfektionsgraden genom att det tillsatta klorets avklingning bestäms för beräkning av Ct-värdet; produkten av kontakttiden och koncentrationen. Genom att jämföra råvattenkvaliteten med avdragen och desinfektions-graden kan en bedömning göras om huruvida reningsprocessen är tillräcklig eller inte. I Mikrobiologisk riskanalys (MRA) bygger man upp en modell av sitt vattenverk i ett program och väljer patogenhalter för råvattnet. Efter det beräknar programmet renings-processens reduktion, riskerna för daglig respektive årlig sannolikhet för infektion samt DALYs, som gör att man kan jämföra risken för vattenburen smitta med exempelvis risken att förolyckas i trafiken. Det amerikanska naturvårdsverket och WHO har satt gränsen för den acceptabla årliga sannolikheten för infektion på grund av vattenrelaterad sjukdom till 1/10 000 invånare respektive 1 µDALYs. Fördelen med MRA är att när man väl byggt upp sitt vattenverk i programmet kan olika scenarion simuleras genom att patogenhalterna varieras. Tyvärr är det svårt att uppskatta patogenhalterna då de provtagningar som skulle behövas sällan är gjorda. Även litteraturvärden kan vara svårt att hitta, särskilt för virus som är så små att de är svåra att analysera. Resultatet av bägge modellerna visade på att Borgs vattenverks reduktionsförmåga är tillräcklig med avseende på bakterier, men inte för virus och parasiter. I MRA är det emellertid svårt att säga hur korrekt detta påstående är, då endast litteraturvärden kunnat användas för de två sistnämnda patogenerna. En del provtagningar har gjorts och då har inga parasiter kunnat påvisas i råvattnet, men för att inte underskatta riskerna användes litteratur-värdena ändå. GDP påvisade inga stora brister i reduceringen av virus och parasiter och i MRA låg resultatet i samma storleksordning som gränsen för DALYs. Eventuellt behöver alltså inga stora åtgärder vidtas för att minska de mikrobiologiska riskerna. De viktigaste stegen i reningsprocessen är långsamfiltrering, desinfektion med fritt klor och fällning och sedimentering med efterföljande filtrering. Infektionsrisken blir störst om fällningssteget slås ut. I samtliga simulerade scenarion låg infektionssannolikheten för bakterier fortfarande på en acceptabel nivå. Det värsta scenariot av de modellerade är om avloppsledningen från Skärblacka skulle börja läcka samtidigt som det regnar kraftigt. Det skulle leda till att förorening från betesmark spolas med ut i Motala Ström där råvattnet hämtas. Detta ger den högsta patogenhalten och därmed också den största infektionsrisken. / Water is one of the most basic things in life and is something we come in contact with on a daily basis. To prevent diseases and infections, the drinking water is purified, mainly in order to reduce the number of pathogens. The most important groups of microorganisms in water purification are bacteria, viruses, and parasites. These groups are very different in many respects and are therefore reduced most efficiently by different purification processes. If more knowledge is gained, the waterworks can optimize the purification process. This would lead to a minimization of the risk of getting infections caused by consuming the drinking water. In this paper two models are used to describe and evaluate Borg’s waterworks, run by Norrköping Vatten AB. Good Disinfection Practice (GDP) is the result of a Norwegian project and is based on formulas and tables. First, the raw water quality is determined by evaluating the presence of microorganisms and the number of people supplied. Then deductions are made due to security precautions and water treatment besides disinfection etc.  Finally the disinfection is determined by calculating the reduction of the added chlorine to gain the Ct-value, which is the product of the contact time and concentration. By comparison of the raw water, the deductions and the disinfection, the purification process can be evaluated as sufficient or not. In Quantitative Microbial Risk Assessment (QMRA) the purification process is modelled and concentrations of pathogens in the raw water are chosen. The program then calculates the reduction of the pathogens by the purification process. The result is also presented as probability of daily or annual infection and DALYs, which makes it possible to compare the risks of waterborne diseases with for example, the risk of traffic accidents. The US Environmental Protection Agency and the World Health Organization have determined the limit for the acceptable annual probability of infection due to water-related disease to 1/10,000 and 1 µDALYs. The advantage of the QMRA is that once you have modelled your purification process a variety of scenarios can be simulated. Unfortunately, it is difficult to estimate the concentrations of pathogens in the raw water and the acquired analysis are rarely made. Even literature values can be difficult to find, especially for viruses due to the difficulties in analysing them because of their small size. Both the models’ results showed that Borg’s waterworks reduction capacity is sufficient for bacteria, but not viruses and parasites. It is however difficult to say how accurate this conclusion is. In QMRA only literature values have been used as a basis to determine the risk for viruses and parasites. In fact, no parasites have been found when samples have been run on the raw water. But since an overestimation of the risk is to be preferred, literature values were used anyway. GDP showed only small shortcomings in the reduction of viruses and parasites and the values obtained from QMRA were in the same order of magnitude as the limit of DALYs. Therefore only small measures might be needed to lower the microbiological risks. The most important steps in the purification process are slow sand filtration, disinfection with free chlorine and precipitation and sedimentation with subsequent filtering. Elimination of the precipitating step results in the greatest risk of infection. In all the scenarios simulated the likelihood of infections caused by bacteria is still acceptable.  The worst-case scenario would be a sewage water leak during heavy raining. The rain would cause excrement from cattle to be washed into and contaminate the raw water in addition to the sewage contamination. This provides the highest concentration of pathogens in the raw water and therefore also the greatest risk of infection.

In this project a LC-MS-method for the analysis of gamma-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, amphetamine and methadone was developed. Initially, the efficiency of the ionisation of the analytes was evaluated with respect to the ionisation technique (ESI, APCI and APPI) and the composition of the mobile phase. In the next step a number of different columns was tested in order to find the one with the greatest potential for separation of the substances in question. Using the selected column, the separation was optimised by means of experimental design and the software The Unscrambler 7.8. The parameters studied were the flow rate, the column temperature and the mobile phase composition. The response variables were the resolution between the target compounds and the retention time of the last eluting compound. These experiments showed that, in order to obtain the best ionisation, the mobile phase should consist of 5 mM formic acid in water and acetonitrile. ESI should be used in the positive mode for all analytes except gamma-hydroxybutyrate, for which the negative mode should be applied. The Hypercarb column exhibited superior retention of the analytes and was therefore selected for further optimisation. The dimensions of this column were 2.1 x 50 mm and the particle size 5 μm, connected to a 2.1 x 10 mm precolumn containing the same packing material. The optimum of the flow rate and the column temperature were 250 μl/min and 20 ºC respectively. For the separation of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol the mobile phase consisted of 100% water with 5 mM formic acid. Thereafter a gradient, up to 70% acetonitrile with 5 mM formic acid, was used in order to elute amphetamine and methadone. Efforts were also made to find an internal standard for the method. However, none of the compounds tested was found suitable. In order to get the method usable for routine analysis, which is the goal, further work is required. A suitable internal standard needs to be added to the method and thereafter work remains with validation of the method.

Liquid chromatography (LC) coupled to mass spectrometry (MS) offers highly selective and sensitive analysis of a wide variety of compounds. However, the use of hyphenated experimental set-ups implies that many parameters may have an effect on the studied response. Therefore, in order to determine optimized experimental conditions it is of vital importance to incorporate systematic procedures during method development. In this thesis, a generic stepwise optimization strategy is proposed that aims at high chromatographic quality, as well as high mass spectrometric response. The procedure comprises (i) screening experiments to identify the most important parameters, (ii) LC studies to ensure sufficient chromatographic separation, (iii) extended infusion experiments in order to maximize precursor signal(s), and in the case of tandem MS (iv) extended infusion experiments to determine optimal conditions for collision induced dissociation and when applicable also ion trap settings. Experimental design and response surface methodology is used throughout the procedure. Further, the general applicability of LC-MS is demonstrated in this thesis. Specifically, a novel quantitative column-switched LC-MS method for ferrichrome, ferrichrysin and ferricrocin determination is presented. Using the method it was shown how the siderophore content varies with depth in podzolic soil profiles in the north and south of Sweden. The parallel approach using LC coupled to both inductively coupled plasma (ICP) mass spectrometry, and electrospray ionization (ESI) tandem MS is also evaluated as a tool to identify unknown siderophores in a sample. Additionally, different trypsin digestion schemes used for LC-ESI-MS peptide mapping were compared. By multivariate data analysis, it was clearly shown that the procedures tested induce differences that are detectable using LC-ESI-MS. Finally, the glutathione S-transferase catalyzed bioactivation of the prodrug azathioprine was verified using LC-MS.

This thesis describes bioanalytical methods for drug determination in biological matrixes, with drugs in focus used against diseases largely affecting low-income countries. Solid-phase extraction is used for sample cleanup, and processed samples are analyzed by liquid chromatography. Developed bioanalytical methods are validated according to international guidelines. Eflornithine (DFMO) is a chiral drug, used for treating human African trypanosomiasis. A bioanalytical method for determination of DFMO enantiomers in plasma is presented. The enantiomers are detected by evaporative light-scattering detection. The method has been applied to determination of D-DFMO and L-DFMO in rats, after intravenous and oral administration of racemic DFMO. It is concluded that DFMO exhibits enantioselective absorption, with the more potent enantiomer L-DFMO being less favored. Sulfadoxine (SD) and sulfamethoxazole (SM) are sulfa-drugs used for malaria and pneumonia respectively. Two methods are described for simultaneous determination of SD and SM in capillary blood sampled on filter paper. The former method allows direct injection of extracts from dried blood spots (DBS), while for the latter method solid-phase extraction is added. Pre-analytical factors contributing to measurement uncertainty is also discussed, and it is concluded that it is of high importance that homogeneity in type of sampling paper and sampling volume is assured. Piperaquine (PQ) is an antimalarial, increasingly used in artemisinin combination therapy. A method for determination of piperaquine in DBS is presented. By using a monolithic LC column, a very short LC analysis of two minutes per sample is achieved. A method for simultaneous determination of three antiretroviral drugs i.e. lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP), in DBS samples is described. The method is applied to drug determination in two subjects after receiving standard antiretroviral treatment. Conclusion is that the method is suitable for determination of 3TC and NVP, and to some extent for AZT.