<p>A new method for analysis of acidic herbicides, mostly phenoxy acids and their esters, in cereals with liquid chromatography-tandem quadrupole mass spectrometry (LS-MS/MS) has been developed. Samples were hydrolyzed with sodium hydroxide in order to release covalently bound compounds followed by neutralization and finally extraction with acidified ethyl acetate. The extraction efficiency for both ester formulations and acids were studied. Acceptable results (70-120 %) were obtained for 2,4-D, dichlorprop, MCPA and mecoprop for both esters and acids. However, low recoveries were observed for ester formulations of dicamba, fluroxypyr, fluazifop and haloxyfop, possibly due to the complex structure of the compounds in combination with the matrix and/or incomplete hydrolysis step. The limit of quantification (LOQ) for targeted pesticides was 0.01 mg/kg. The method has been tested in the EU Proficiency Test for cereals with good results.</p>

<p>A synthetic pathway towards the cap-structure of 2,2,7-trimethylguanosine containing a methylene modified triphosphate bridge have been investigated. The modification to the triphosphate bridge is hoped to slow down cap degradation and give the connected  oligunucleotide an increased lifetime. This could result in an better understanding of nuclear transport of oligonucleotides and could thereby helping to develop new treatments for different diseases. The synthesis relies on a coupling reaction between the 2,2,7-trimethylguanosine 5’phosphate and 2’-<em>O</em>-methyladenosine with a 5’-pyrophosphate where the central oxygen has been replaced by a methylene group. The reaction pathway consists of 9 steps of which 8 steps have been successfully performed. The last step, which includes a coupling reaction, was attempted but without successful identification and isolation of the cap-structure, and will need further attention. The reaction has been performed in a milligram scale with various yields.</p> / Presentation utförd

<p>Traditionally, rolling mills use emulsions based on a mixture of oil and water for lubrication. Since two years ago SAPA has been using (instead of oil) a synthetic lubricant so called conditional emulsion for hot-rolling of aluminum. This lubricant is water based and homogenous at ambient temperature, but switches to a two-phase system at heating above the cloud point.</p><p>This project aims to validate and if necessary modify an existing HPLC method for quantifying two out of three key components (A, B and C) in the conditional emulsion. Attempts to develop a method to quantify the pH adjusting components, X and Y were also made. These two methods are required to optimize the lubricant.</p><p>Due to the complexity of the components, it has been difficult to present a method for quantification, and HPLC with ELS detection was chosen after a long series of trials. Due to a few uncontrollable parameters the proposed analysis method has tendencies to be unstable. The column used is sensitive to changes in equilibrium and ELSD is also less sensitive and less reproducible than the commonly used UV-detector.</p><p>While the proposed assay method shows somewhat large relative standard deviations the method has been shown to produce sufficiently precise and accurate data for the intended purpose.</p><p>Development of a method for the pH-adjusting components X and Y was more difficult than expected. For some reason their difference in chemical properties does not show satisfying impact in the chromatograms.</p><p>This method is still in its cradle and needs further development.</p><p> </p>

<p>Immobilization of different bio- and organic molecules on solid supports is fundamental within many areas of science. Sometimes, it is desirable to obtain a directed orientation of the molecule in the immobilized state. In this thesis, the copper (I) catalyzed Huisgen 1,3-dipolar cycloaddition, referred to as a “click chemistry” reaction, was explored as a means to perform directed immobilization of small molecule ligands on gold surfaces. The aim was to synthesize alkyne- and azide-terminated alkanethiols that would form well-organized self assembled monolayers (SAMs) on gold from the commercially available substances orthoethylene glycol and bromo alkanoic acid. N-(23-azido-3,6,9,12,15,18,21-heptaoxatricosyl)-n-mercaptododekanamide/hexadecaneamide (n = 12, 16) were successfully synthesized and allowed to form SAMs of different compositions to study how the differences in density of the functional groups on the surface would influence the structure of the monolayer and the click chemistry reaction. The surfaces were characterized by different optical methods: ellipsometry, contact angle goniometry and infrared reflection-absorption spectroscopy (IRAS). The click reaction was found to proceed at very high yields on all investigated surfaces. Finally, the biomolecular interaction between a ligand immobilized by click chemistry on the gold surfaces and a model protein (bovine carbonic anhydrase) was demonstrated by surface plasmon resonance using a Biacore system.</p>

<p>The insulin-like 3 peptide is autocrine/paracrine insulin-related hormone with a size of approximately 6kDa [1]. It mediates through a leucine richG-coupled receptor named LGR8. INSL3 is mainly expressed in human Leydig cells and is directly responsible for migration of the testis during the pre-natal period in maledevelopment. [2]</p><p>INSL3 mRNA has recently been verified in human platelets whereas no mRNA has been detected for LGR8 (by Sanofi-Aventis GmbH in Frankfurt,Germany), indicating that INSL3 might be released through paracrine functions at sites of platelet adhesion and aggregation upon a vascular injury.Furthermore, has activated platelets been shown to translate essential proteins upon activation, in a term called “signal-dependent protein synthesis”.The B-Cell lymphoma-3 protein (BCL-3) is an example of such a protein [3], and there is a possibility that INSL3 might be also.</p><p>In this thesis we wanted to detect the relaxin- like peptide 3 hormone (INSL3). (Its function, location and the timeframe of its release, when/if it issecreated in stimulated platelets).The source of platelet-derived INSL3 can be found with Western blotting and Enzyme immunoassay.</p><p>Detection of the insulin-like 3 peptide in human platelets turned out to be a difficult challenge due to the small amount of INSL3 secretion uponplatelet activation; hence the total amount of INSL3 produced might be below detection limit.</p>

<p>Platelets are important for the healing of damaged blood vessels since they have an importantpart to play in the coagulation process. At the same time, the blood must be kept fluid and notcoagulate at the wrong time. Therefore there are factors that effect the aggregation of plateletsin a positive or a negative way.</p><p>Previous investigations have shown that platelets during stirring conditions produce reactiveoxygen species (ROS) that weaken the inhibiting effect of nitric oxides (NO) on platelets andthat the drug Dexamethasone (Dex) can reduce the ROS-production.</p><p>The aim of this project was to investigate if glucocorticoids, in this case Dexamethasone,could restore the inhibiting effect of NO on platelets and if there was any decrease in ROS-production.</p><p>The result of the ROS-measurements showed a great variance and it was difficult to draw anyconclusions from them, but a clear decrease in ROS, as previous reported, was not shown. In the aggregation experiments the inhibiting effect of NO was observed through the drug S-nitroso-N-acetylpenicillamine (SNAP), a NO-donator.</p><p>From the aggregation experiments, the result seemed to be that SNAP during longerincubation time lost its inhibiting effect, probably because the cells become desensitized.With superoxid dismutase (SOD), the effect of SNAP increased, both in the experiment withlonger and shorter incubation times. Dex seemed to reinforce the aggregation in relation toboth SOD and SNAP. To understand this relation further, more investigations must be done.Another interesting experiment would be to do combinations of experiments monitoring bothaggregation and ROS-production at the same time.</p> / <p>Trombocyterna, blodplättarna i blodet, är livsviktiga för att människor inte ska förblöda vid enskada. Samtidigt måste blodet hållas flytande och inte koagulera i onödan och därför finns deti kroppen en mängd faktorer som verkar pro- eller antiaggregerande.</p><p>Tidigare undersökningar har visat på att trombocyter har en omrörningsberoendesyreradikalproduktion (ROS) som försvagar kväveoxids (NO) antiaggregerande effekt och attläkemedlet Dexametason (Dex) kan minska denna produktion.</p><p>Detta projekt syftade till att ytterligare studera om glukokortikoider, i detta fall Dexametason,kunde återställa NO:s effekt på trombocyterna och om de i någon grad minskaderadikalproduktionen.</p><p>Resultatet av ROS-mätningarna blev väldigt varierande och svårtolkade och några säkraslutsatser kunde inte dras, men en tydlig minskning i produktionen som tidigare observeratskunde inte upptäckas. I aggregationsförsöken observerades NO:s inhibitoriska verkan genomS-nitroso-N-acetylpenicillamine (SNAP), en NO-donator.Resultaten tyder på att SNAP under en längre inkuberingstid tappar sin inhiberande förmågapå trombocyterna, vilket förmodligen beror på att cellerna desensibiliseras.Superoxiddismutas (SOD) verkar ha en förstärkande effekt på SNAP oavsett ominkuberingstiden innan dosresponstillsats av trombin är lång eller kort, medan Dex tenderaratt förstärka aggregeringen både i förhållande till SNAP och SOD. För att få mer klarhet omdessa resultat är korrekta måste fler upprepningar göras och dessutom borde man genomförakombinationsförsök där man samtidigt övervakar ROS-produktion och aggregering.</p>

<p>There is convincing research which shows that the enzyme alkaline phosphatase (ALP) has a central role in the mineralization of bone, more precisely that its catalytic activity is needed in the process. ALP is found on the surface of matrix vesicles where the mineral is formed. One theory about the function of the enzyme is that it binds to fibrous collagen in the bone and thereby incorporating the mineral into the bone. The purpose of this study is to establish whether ALP binds to collagen. If this is the case, more elaborate studies around this will be performed. The strength of the binding between collagen and the different types of ALP will be evaluated, as well as on which part of the collagen the binding occurs. The binding is going to be studied by constructing a method for the ÄKTApurifier system.</p><p> </p><p>Initially, the pureness of the different type of collagens was determined by using SDS-PAGE and the activity of the different types of ALP was established. These were also compared with a native PAGE. In SDS-PAGE, bovine type I collagen showed markings for a triple helix, a double helix and two single strains, α₁ and α₂. Bovine type II collagen showed markings for a double helix and α₁-strains. Human type I collagen showed markings for a triple helix, two double helixes, two α-strains and contaminations. Trials with collagen in Native PAGE did not provide any results. However, the trials with ALP revealed that the different types of ALP had different charge.</p><p> </p><p>Thereafter, blotting was performed. The results showed that all the different types of ALP, besides from E. coli, binds to bovine collagen type I and II and human collagen type I, however within various periods of time. In the trials with collagen coated plates the acquired results showed that some of the different types of ALP bind to collagen. ALP from liver binds the strongest to both collagen type I from rat and type IV from mouse. Intestinal ALP also binds to both types of collagen but not nearly as strong as liver ALP. Serum from rats did bind to collagen type I from rat but not to collagen type IV from mouse. ALP from kidney and human serum did not bind to either types of collagen. The trials concerning the ÄKTApurifier system were executed with ALP from liver alone because it had been proven to bind to bovine type I collagen through the previous methods. The results confirmed that ALP from liver binds to this type of collagen.</p><p> </p><p>The conclusions from this study are that ALP does indeed bind to collagen and does so to the triple helix and double helix form as well as the single strains of collagen. In other words the part of the structure in collagen that ALP binds to must exist in all three stages of collagen formation. Furthermore, it seems like some of the different types of ALP has a higher affinity for binding to collagen, as the time for binding to collagen varies for the different types of ALP. The results differed between methods concerning different types of ALP. Although, the method we consider to give the best result was blotting. However, the method using ÄKTApurifier can be complementary but needs further development.</p>

<p>The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%.</p><p>The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.</p>

<p>The main purpose of the project was to identify potential environmentally harmful substances which can be leached from rubber asphalt. A method for analysing asphalt was developed and three rubber asphalt materials were analysed after being cryogrinded. One of the materials was also tested in a road machine made for testing of asphalt paving. The particles created in the machine were analysed in the same manner as the cryogrinded asphalt materials.</p><p>The asphalt materials were leached by water during 24 hours. The leachates were extracted with dichloromethane, dried with sodium sulphate and concentrated to a small volume. The extracts were analysed with gas chromatography-mass spectrometry (GC-MS). Due to low concentrations of substances the GC-MS was operated in SIM-mode (Selected Ion Monitoring). Thirteen substances were chosen for the analysis. The substances were aniline, benzothiazole, butyl benzyl phthalate, bisphenol-A, decanoic acid, dibutyl phthalate, diethylhexyl phthalat, phenanthrene, chrysene, naphthalene, 4-n-nonyl phenol and 4-tert-octyl phenol. Benzothiazole and 4-tert-octyl phenol were detected and quantified in the leachates.</p><p>In addition to the analysis of organic substances, pH was measured too. The leachates produced in this project were also sent to an analysis company for several analyses, for example analysis of metals and sulphur. Toxicity tests were performed on the same leachates within an exam work made by Gro Runeman at Lund University. The results of the metal analysis, sulphur analysis, and toxicity tests are not covered within the scope of this report. For the results of these tests and analyses, see Gro Runeman’s report: <em>Evaluating toxicity of asphalt leachates</em>.</p> / <p>Detta examensarbete är utfört vid Statens Geotekniska Institut (SGI). Huvudsyftet med arbetet är att identifiera potentiellt miljöfarliga ämnen som kan laka ut från gummiasfalt. En metod för analys av asfalt har tagits fram och tre olika gummiasfalter har analyserats efter att ha kryomalts. En av dessa asfalter har också använts i en provvägmaskin där det damm som bildades har samlats upp och analyserats. Inom ramen för examensarbetet har också en litteraturstudie gjorts för att bland annat ta reda på vad som är gjort inom området sedan tidigare.</p><p>För att ta reda på vad asfalten innehåller för föreningar gjordes först en fastfasextraktion av krossad (ej kryomald) asfalt där diklormetan användes som lösningsmedel. Från början var tanken att en GC/MS-screening skulle göras för att på så sätt få en överblick av samtliga organiska ämnen som finns i asfalten men på grund av de väldigt låga halterna av i gaskromatografen analyserbara föreningar var det nödvändigt att begränsa analysen till några få föreningar. De föreningar som analysen inriktade sig mot var anilin, bensotiazol, bensylbutylftalat, bisfenol-A, dekansyra, dibutylftalat, di(etylhexyl)ftalat, fenantren, krysen, naftalen, 4-n-nonylfenol, pentaklortiofenol och 4-tert-oktylfenol.</p><p>Laktester utfördes genom att de kryomalda asfaltmaterialen lakades med vatten under 24 timmar. Efter extraktion, torkning och koncentrering analyserades lakextrakten med avseende på de föreningar som hittats vid fastfasextraktionen. I lakvattnen hittades bensotiazol samt 4-tert-oktylfenol. Dessa föreningar kvantifierades genom att en enpunktskalibrering gjordes.</p><p>Utöver analyserna ovan mättes även pH på lakvattnen. Lakvattnen skickades också på metall- och svavelanalys samt turbiditets- och fenolindexmätning till ett större analysföretag. Toxicitetstester har utförts på samma lakvatten av Gro Runeman inom ramen för ett examensarbete vid Lunds Universitet. För resultaten från toxicitetstester, metallanalyser samt turbiditets- och fenolindexmätningar hänvisas till Gro Runemans rapport: <em>Evaluating toxicity of asphalt leachates</em>. Delar av denna rapport är skrivna i samarbete med Gro Runeman.</p>

<p>Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum.</p><p>The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with <em>tert</em>-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.</p>