Introduction: Post-mortem whole blood samples differ greatly in quality, lipemia is one cause of concern in toxicological analyses. Around 4 % of all samples sent to RMV are given a notation of lipemic content. The aim of the thesis was to study the effects of lipemia on the quantification of 14 benzodiazepines and 5 similar sedative and antianxiety drugs as well as evaluate the pre-analytical process aiming to reduce the effects of lipemia.  Methods: Blood samples were simulated with bovine blood, analyte spiking, and lipid spiking with either the nutrition emulsion Intralipid or with a mixture of post-mortem lipids from authentic samples. The outset was the by RMV currently used LLE method followed by UPLC- MS/MS and the extraction method was altered and evaluated. Matrix effects were also studied.  Results: Lipemia were found to be a great interference when quantifying benzodiazepines. For most analytes, internal standard could compensate for the loss of analyte but there was a problem with analytes not having their own IS. The 7-amino-compounds were greatly affected by lipemia and propiomazine and dihydropropiomazine showed extreme losses. Equilibration of IS did not result in similar loss as analyte. Dilution of sample reduced losses caused by lipemic content. SPE resulted in extracts free from lipids and high yields but there were analyte losses similar to LLE. No matrix effects from the lipids were found. Samples spiked with Intralipid gave poorer analyte yields than those spiked with post-mortem lipids.  Conclusion: Dilution is the most successful method to reduce pre-analytical matrix effects as long as the concentration is not so low that it risks getting lower than the analytical limits when doing so. Not homogenising samples before sampling is giving incorrect results. SPE could, if optimised for the analyte retention and elution, remove lipids from samples and obtain accurate analyte concentrations. Pooling lipids from post-mortem samples is a possible method for simulating lipemic whole blood. Intralipid and the PM-mix gave the same indications, but to different extents. Further studies where the ability to mimic authentic lipids are needed for both Intralipid and PM-mix.

Although Lake Victoria is threatened by pollution there is a lack of knowledge about pharmaceuticals contaminants drained into the lake from large cities bordering the lake. Hence, the purpose of this project was to develop, validate and apply a method for analysis of pharmaceutical compounds accumulating in the Lake Victoria sediments. A simple quantitative method for 11 pharmaceuticals combining accelerated solvent extraction, solid phase extraction, trimethylsilylation derivatization, and gas chromatography mass spectrometry was developed, partly validated, and applied to 18 surface sediments and a sediment core dated using the 210Pb method. The results showed the presence of the pharmaceuticals estriol, gemfibrozil, metoprolol, ketoprofen, naproxen, 17α-ethinylestradiol and estrone concentrated around the regions Napoleon Gulf and Thurston Bay with accumulation rates decreasing towards the top of the sediment core. Nonetheless, a randomness in the distribution of these compounds behooves a systematic assessment investigating not only the provenance of these compounds but also further investigations to errors meaning that this study should be treated as a preliminary investigation.

The application of different analytical techniques is fundamental in forensic drug analysis. In the wake of the occurrence of large numbers of new psychoactive substances possessing similar chemical structures as already known ones, focus has been placed on applied criteria for their univocal identification. These criteria vary, obviously, depending on the applied technique and analytical approach. However, when two or more substances are proven to have similar analytical properties, these criteria no longer apply, which imply that complementary techniques have to be used in their differentiation. This work describes the synthesis of some structural analogues to synthetic cannabinoids and cathinones based on the evolving patterns in the illicit drug market. Six synthetic cannabinoids and six synthetic cathinones were synthesized, that, at the time for this study, were not as yet found in drug seizures. Further, a selection of their spectroscopic data is compared to those of already existing analogues; mainly isomers and homologues. The applied techniques were mass spectrometry (MS), Fourier transformed infrared (FTIR, gas phase) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. In total, 59 different compounds were analyzed with the  selected techniques. The results from comparison of spectroscopic data showed that isomeric substances may in some cases be difficult to unambiguously identify based only on their GC-MS EI spectra. On the other hand, GC-FTIR demonstrated more distinguishable spectra. The spectra for the homologous compounds showed however, that the GC-FTIR technique was less successful compared to GC-MS. Also a pronounced fragmentation pattern for some of the cathinones was found. In conclusion, this thesis highlights the importance of using complementary techniques for the univocal identification of synthetic cannabinoids and cathinones. By increasing the number of analogues investigated, the more may be learnt about the capabilities of different techniques for structural differentiations, and thereby providing important identification criteria leading to trustworthy forensic evidence.

Exosomer är extracellulära vesiklar i nanostorlek som frigörs från cellerna till den extracellulära matrisen. Exosomer är laddade med nukleinsyror, proteiner, och lipider, och fungerar som kommunikatorer mellan celler. Det har gjort dem mycket attraktiva for forskning inom terapi, diagnostik och transport av läkemedel. För att använda exosomer i kliniska tillämpningar behöver standardiserade metoder för isolering, rening och analys av exosomer att utvecklas.  Detta projekt syftar till att sätta upp en snabb metod som använder "multiangle light scattering" (MALS) i kombination med kromatografi for att bestämma storleken på exosomer. Olika storlekskromatografi (SEC)/jonbyte (IEX)-kolonner kommer att undersökas och användas for analys av storlekar på exosomer. Partikelstorleken som erhålls från MALS kommer sedan att verifieras med nanoparticle tracking analysis (NTA).  För SEC-MALS-analyserna eluerades exosomerna i dödvolymen. IEX-MALS-metoden separerade exosomerna enligt resultatet. Exosomer i storlek mellan 60-110 nm eluerades ut med 800 mM NaCl. Större exosomer och eventuella aggregat i storlek 120-200 nm eluerades ut med 1200 mN NaCl.  Resultatet visar att signalen från MALS indikerar var exosomer i kromatogrammen elueras ut och kan ge värdefull information om storleksfördelningen i en topp. SEC-MALS-resultatet är dock inte reproducerbart, eftersom det ibland sker en förändring i storleksfördelningen över toppen. Resultatet har också visat att arean for toppen i dödvolymen varierar mellan körningarna, vilket förmodligen orsakats av att exosomer interagerar med den stationära fasen eller filtret i kolonnen. Förskjutningen i storleksfördelning observerades också i IEX­MALS-metoden. Medelvärdet av partikelstorlekarna som beräknades från SEC-MALS överensstämmer med storlekarna beräknade med NTA. SEC-MALS-metoden behöver förbättras för att fa reproducerbara resultat. Den beräknade storleken från IEX-MALS stämde inte överens med storleken från NT A. Jonbytesanalysen skulle kunna upprepas och fraktioner skulle kunna analyseras med andra tekniker för att verifiera resultatet i framtida arbete. Effekten av den höga saltkoncentrationen på exosomerna behöver också undersökas ytterligare. / Exosomes are nanosized extracellular vesicles released from the cells into the extracellular space. Exosomes are loaded with nucleic acids, proteins, and lipids, and work as communicators among cells. This has made them very attractive for research in therapeutics, diagnostics and drug delivery applications. Standardised exosome isolation, purification, and analysis methods need to be developed to use exosomes in clinical applications.  This project aimed to set up a quick method using multiangle light scattering (MALS) combined with chromatography techniques to determine exosome sizes. Different size exclusion (SEC)/ion exchange (IEX) columns will be investigated and used to analyse exosome sizes. The particle size obtained from MALS will then be verified with nanoparticle tracking analysis (NTA).  For the SEC-MALS analysis, the exosomes were eluted in the void volume. The IEX-MALS method separated the exosome sample. The exosome eluted with 800 mM NaCl ranged between 60-110 nm in diameter. The exosomes eluted with 1200 mM NaCl ranged between 120-200 nm in diameter. The result shows that the light scattering intensity from MALS indicates where the exosomes elute in the chromatograms and gives valuable information about the size distribution in a peak. However, the SEC-MALS result is not reproducible, as sometimes, a shift in the size distribution over the peak occurs.  The result has also shown that the void peak area varies between the runs, caused mainly by the exosomes interacting with the resin or the column's filter. The shift in size distribution was also observed in the IEX-MALS method.  The average sizes calculated from SEC-MALS agree with the sizes calculated with NTA. The SEC-MALS method needs to be improved to obtain reproducible results. The calculated size from IEX-MALS did not agree with the size from NTA. The ion exchange analysis could be repeated and further analysed with other techniques to verify the result in future work. The effect of the high salt concentration on the exosomes also needs to be further investigated.

This study focuses on a general method that has been developed for the identification of both polar- and nonpolar pesticidespolar pesticides in a known solution from EPA 8151 Herbicide acid mix by Merck including the ordered non-polar pesticide Prosulfocarb. The EPA-solution contains 16 analytes that has been completely identified when derivatized and spiked in acetone. The solution has also been spiked in blood samples, resulting in five calibration solutions in the range of 0,01 – 2,5 µg/g, followed by quantification. Identification of dinoseb and bentazone, spiked in blood, failed whereas the remaining 145 analytes were identified. The method uses headspace (HS) solid phase microextraction (SPME) in combination with gas chromatography (GC) with mass spectrometry (MS) in both scan and selected ion monitoring mode. A standard extraction- and derivatization procedure has been performed for the analysis of alcohols, phenols, and carboxylic acids with help from a protocol regarding the analysis of ethylene glycol in blood. Samples were introduced on the column with splitless injection where 1 µl were injected with an injector temperature of 250oC. Effective separations were achieved by using GS-GasPro PLOT-column (30 m x i.d. 320 µm x df 0 µm) in combination with a temperature programme that started at an initial temperature of 80oC (1 min) that increased by 10oC/min up to 280oC (1 min). The limit of detection (LOD) for the pesticides, spiked blood, were 0,01 – 2,5 µg/g where the lowest limit of 0,01 µg/g meant difficult identification whereas a greater identification was made at 0,05 µg/g. No identification was succeeded for the most polar substances in the forms of amines and amides in combination with carboxylic acid. Identification was however made for the less polar pesticides in the forms of alcohols, phenols, and carboxylic acids. The method must be further developed to identify the highly polar pesticides in different chemical classes. The current method can be used in occurring intoxications and in autopsy cases.

In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers. All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width. In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls. In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting <100> crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls. For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis. / QC 20101108

Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sin tur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLC-MS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering. / Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind to proteins in the blood, but only unbound drug can diffuse over capillary membranes and bind to the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ blood samples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common in critically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation.

Supercritical fluid chromatography (SFC) is an expanding chromatographic technique that is part of the evolving field of green chemistry due to its short run times and use of non-toxic solvents in combination with recycled CO2. The use of a suitable column is essential for separation science in general and there is a wide variety of SFC compatible columns available on the market. In collaboration with the AstraZeneca Gothenburg Separation Science Laboratory (SSL), an investigation of 25 stationary phases in combination with four different mobile phases was conducted in order to evaluate interactions between the mobile phase, stationary phase and analytes. The aim was to find a new column set, able to operate with a wide range of molecules at both analytical and preparative scale. The 36 compounds analysed were chosen to represent the chemical space suitable for current and foreseen compounds of interest within AstraZeneca. Furthermore, to mitigate the risk of degradation of sensitive compounds in basic conditions, different combinations of basic stationary phases and neutral mobile phases were investigated.The study was performed using a Waters Acquity UPC2 system equipped with a photodiode array (PDA) detector and a single quadrupole detector (SQD). The data was gathered by Empower 3 and analysed using statistical methods to evaluate corelations and orthogonality with the help of Python. Visualisations were produced using Pandas, XLstat and SIMCA.The results evaluated retention time, symmetry factor, distribution of retention times of compounds over the columns, and DMSO retention. The results showed that MeOH and the basic additive NH3 included in the mobile phase composition provided the shortest retention times, best peak symmetry and distribution of compounds. It was concluded that Kromasil 2EP, Kromasil Diol, DC Pak PBT, and Kromasil NH2 columns represent a diverse set for the intended chemical space. They all also operate with good results without additive, for the compounds used. / Superkritisk vätskekromatografi (SFC) är en växande kromatografisk teknik som tack vare sina snabba analyser, användandet av icke toxiska lösningsmedel, samt återvinningsbar CO2 utgör en del av den allt mer populära “gröna” kemin. Det finns en mängd kolonner utvecklade för SFC på marknaden, och att använda rätt sorts stationär fas är en viktig del för separationen av olika ämnen. I samarbete med Separation Science Laboratory på AstraZeneca i Göteborg, har 25 olika stationära faser i kombination med fyra mobila faser studerats för att analysera interaktionerna mellan faserna och analyterna. Målet är att hitta en uppsättning av kolonner som kan användas vid upprening av substanser med en stor variation av kemiska egenskaper/deskriptorer. För den här studien har 36 molekyler som representerar den kemiska rymd som AstraZeneca jobbar i använts. För att minimera risken av nedbrytning av känsliga molekyler i basiska miljöer, är även basiska kolonner med neutrala mobilfaser inkluderade. Under studien användes SFC systemet Acquity UPC2, utrustad med en Diode array detektor, en Single quadrapole detektor samt Empower 3 för insamling av data. Den statistiska analysen utfördes i Python och visualiseringen gjordes med hjälp av Pandas, Xlstat och Simca.Resultaten utvärderades med avseende på retentionstid, symmetrifaktorn, fördelningen av substanser över kolonnerna och retention av DMSO. Resultaten visade att MeOH med NH3 som additiv i mobilfasen gav kortast retentionstid, bäst symmetri och bredast fördelning av substanser. Slutsatserna var att kolonnerna Kromasil 2EP, Kromasil Diol, DC Pak PBT, and Kromasil NH2 tillsammans representerar den diversa uppsättning av kolonner som passar den kemiska rymnd som användes i studien. Samtliga kolonner kan även köras med goda resultat i neutrala mobila faser, med de betingelser som använts.

The adsorption isotherm of two weak bases, Promethazine hydrochloride and Propranolol hydrochloride, were determined with isocratic reversed-phase liquid chromatography, with a 60 w% methanol in 20 mM sodium acetate buffer pH 4 as the mobile phase, and calculated by the elution by characteristic points method. The data obtained from the method were then fitted into the Langmuir isotherm and the electrostatically modified Langmuir. Propranolol fitted reasonably good into the models while Promethazine was not as good. When Promethazine and Propranolol were together in the same sample, there was indication of competition of the adsorption sites. For comparing retention and peak shape between a C18 column and a mixed mode column, Waters XBridge C18 and Thermo Scientific Acclaim WCX-1, were tested in gradient elution with 11.32 mM sodium acetate buffer and 10–70 % methanol. The mixed-mode column gave significantly better peak shapes, while the retention time were longer compared to the C18 column. / Adsorptions-isotermerna för två svaga baser, Prometazin hydroklorid och Propranolol hydroklorid, bestämdes med isokratisk omvänd-fas vätskekromatografi, med w% 60 metanol i en 20 mM natriumacetatbuffert pH 4 som mobil fas, och beräknad med metoden elution by characteristic points.  Från metoden erhållna data passades till Langmuir isotherm och den elektrostatiskt modifierade Langmuir. Propanolen passade ganska bra till de olika isotermerna, medan Prometazin var något sämre passad. När Prometazin och Propranolol var tillsammans i samma prov, fanns det indikationer på konkurrens om adsorptionsställen. För jämförelse av topparnas form och retentionstid mellan en C18-kolonn och en mixed-mode-kolonn, användes Waters XBridge C18 och Thermo Scientific Acclaim WCX-1, som testades i gradient eluering med 11, 32 mM natriumacetatbuffert och 10–70 % metanol. Mixed-mode-kolonnen gav åtskilligt bättre toppar, medan retentionstiden var längre jämfört med C18-kolonnen.

Oligonucleotides (ONs) are widely used in different applications in life science, forensic, in i.e. family tree DNA test for humans and in diagnostic applications in several fields. The use of ONs in biopharmaceutical therapeutic areas also generates new challenges handling more complex molecules which results in the need of further developed analytical techniques. Anion exchange chromatography (AEX) is a common separation technique for biomolecules and is based on charge attraction between the analyte and the stationary phase. The chromatographic system is complex and often high pH and a high salt concentration is needed for the elution to occur, which in some systems can be corrosive for both the column and the instrument. The aim of this study was to evaluate new mobile phase compositions with lower salt concentrations, organic modifier, and usage of a buffer to increase the control of the pH. This was done by evaluation of three columns developed for AEX and uses different chemical and methodical modification of the mobile phase to control the retention to the stationary phase. The influence of pH, temperature, and methanol (MeOH) content in the buffer were studied by evaluation of resolution, asymmetry, and efficiency responses. Three oligonucleotides with 16, 18 and 19 T-bases in the chain were used in the study of three AIE columns. High pH, elevated temperature and the addition of an organic modifier were used for unfolding of the oligonucleotide chain and generating more efficient separations. Other parameters such as gradient slope and initial concentration of the eluting buffer were also studied, and the findings clearly show that the chromatographic conditions influence the resolution, asymmetry, and efficiency. / Oligonukleotider används i flera olika branscher som forensik och inom life sience till diagnostiska och medicinska applikationer. Eftersom applikationsområdena ökar och forskningen går framåt så blir molekylerna mer komplexa, vilket i sin tur kräver att dom analytiska teknikerna också behöver utvecklas. En vanlig separationsteknik för biomolekyler är anjonbyteskromatografi, då den bygger på laddningsattraktion mellan analyten och den stationära fasen. Oligonukleotider har en negativ nettoladdning på grund av den negativt laddade fosfatgruppen i kedjan. Genom att modifiera de kemiska egenskaperna i den mobila fasen är det därför möjligt att i viss grad kontrollera retentionen till den stationära fasen.       Då det kromatografiska systemet är komplext och det ofta behövs högt pH och/eller en hög saltkoncentration för eluering av analyten kan det i vissa system verka korrosivt för både kolonnen och instrumentet. Det initiala målet med detta arbete var att ta fram olika mobila faser med lägre saltkoncentration, organiskt lösningsmedel och användande av buffer för att kontrollera pH. Detta gjordes genom att utvärdera den kromatografiska kapaciteten hos tre olika anjonbyteskolonner samt använda olika kemiska- och metodiska modifieringar av den mobila fasen för att kontrollera analytens retention. Mer specifikt så kommer påverkan av pH, temperatur och innehåll av metanol i den mobila fasen att studeras genom att utvärdera upplösning, asymmetri och effektivitet i de erhållna kromatogrammen. Analytmixen innehåller tre olika oligonukleotider vilka består av 16, 18 eller 19 T-baser i sekvensen. Genom att höja pH, temperatur och innehåll av metanol i den mobila fasen påvisas mer effektiva separationer. Andra faktorer som gradientlutning och initialkoncentration av den eluerande fasen studeras också med goda resultat när det gäller dess påverkan på upplösning, asymmetri och effektivitet.