Dietary intake is one of the major routes of human exposure to perfluoroalkyl and/or polyfluoroalkyl substances (PFAS). The objective of this study was to measure perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexane sulfonic acid (PFHxS) in organic and conventional egg from Sweden (n=8, consisted of 4 pooled eggs and 4 individuals) and China (n=9, consisted of 4 pooled eggs and 5 individuals) and compare the concentrations of PFAS between the two categories (organic and conventional). Also, to evaluate if there was any difference in concentrations of PFAS between both countries. In the end, evaluation of tolerable weekly intake of PFOS and PFOA due to consumption of egg recommended by the European Food Safety Authority (EFSA) was conducted if consuming these eggs would cause any human health risk. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to analyze PFOS, PFOA and PFHxS in the egg samples. In egg samples from China, PFOA was the predominant PFAS; in an organic egg sample from Shenzhen with concentration up to 2000 pg/g, making up to 86% of the 3 PFAS. In contrast, PFOS had the greatest concentration of all PFAS in egg samples from Sweden and was detected in organic egg sample with concentration up to 184 pg/g, making up to 78% of the 3 PFAS. PFOA in samples from China was 18 times higher compared to egg samples to Sweden; results showed no significant differences in PFAS concentrations in egg samples between Sweden and China. In samples from China, concentrations of PFAS had total mean of 50 pg/g for PFOS, 373 pg/g for PFOA and 13 pg/g for PFHxS. In Sweden, mean concentrations of PFOS, PFOA, and PFHxS were found to be 5, 2, and 1,5 times (respectively) higher in organic eggs when compared to conventional. However, significant difference was only observed for PFOS in Swedish organic eggs (p<0.05, t-7.96, df=6). The different concentrations of contamination between organic and conventional egg could be due to the fish powder in organic chicken feed and ingestion of soil through pecking. The result suggests that current concentrations of PFOS and PFOA in organic and conventional chicken eggs are unlikely to cause any immediate harm to Swedish populations. For Chinese population since the consumption of egg has a high risk of exceeding the TWI, the current concentration of PFOA in organic chicken eggs may cause harm to the population based on TWIs established by EFSA. Further investigation is needed with more samples to be analyzed to confirm this point.

Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were

Mit der vorliegenden Studie sollte zu dem wichtigen Forschungsfeld der Pharmakogenetik beigetragen werden, indem zum einen eine einfache und sichere kombinierte Phänotypisierung der drei zuvor erwähnten CYPs (CYP2D6, CYP2C9 und CYP2C19) entwickelt, und zum anderen die Vorhersagekraft des Genotyps für den gemessenen Phänotyp näher untersucht werden sollte. Es ist uns gelungen eine sichere, einfache, schnelle und kombinierte Phänotypisierung der beiden wichtigen Monooxygenasen CYP2D6 und CYP2C9 zu etablieren. Zunächst wurden dazu Wechselwirkungsstudien mit den ausgewählten Testsubstanzen Dextromethorphan (DEX, CYP2D6), Flurbiprofen (FLB, CYP2C9) und Omeprazole (OME, CYP2C19) durchgeführt. Es konnte gezeigt werden, dass DEX und FLB als Kombination verabreicht werden können. Die Gabe von OME gemeinsam mit FLB verändert jedoch das Ergebnis der CYP2C9 Phänotypisierung. Dies ist eine neue Erkenntnis, denn noch 2004 wurde ein Phänotypisierungscocktail veröffentlicht, der die Kombination von FLB und OME enthielt. Bei der genannten Studie wurden jedoch, unseres Wissens nach, keine Wechselwirkungsstudien zu den einzelnen Testsubstanz-Kombinationen durchgeführt. Die von uns entwickelte Phänotypisierungsmethode wurde durch Wechselwirkungsstudien verifiziert. Sie ist jedoch auch in anderen Bereichen den bisher veröffentlichten phänotypisierungscocktails überlegen. Zum einen wurden nur sehr kleine Dosen sicherer Testsubstanzen verwendet. Dies wurde durch Entwicklung neuer, sensitiver LC-MS/MS Methoden ermöglicht. Zum anderen ist diese neue Prozedur schnell und nicht-invasiv durchführbar. Nach Verabreichung der Testsubstanz muss der Urin nur für zwei Stunden gesammelt werden. Zudem weisen unsere Ergebnisse darauf hin, dass die normalerweise durchgeführte, aufwendige Glucuronidspaltung des CYP2D6 abhängigen DEX-Metaboliten, Dextrorphan, vermutlich vernachlässigt werden kann. Die wichtigsten Ergebnisse dieser Studie sind jedoch die Einblicke, die in die Vorhersagekraft der CYP2D6 und CYP2C9 Genotypen für die entsprechenden Phänotypen gewonnen werden konnten. Fast 300 phänotypisierte Kaukasier wurden auch in Hinsicht auf die wichtigsten varianten Allele von CYP2D6, CYP2C9 und CYP2C19 mithilfe bekannter und neu etablierter Methoden genotypisiert. Aufgrund der parallelen Phäno- und Genotypisierung konnten Geno- und Phänotyp direkt korreliert werden. Mit linearen Modellen war es möglich, allen detektierten varianten CYP2D6- und CYP2C9-Allelen Aktivitätskoeffizienten zuzuweisen. Diese können nun verwendet werden, um den Beitrag der einzelnen Allele zur resultierenden Enzymaktivität zu bestimmen, wodurch sich die Vorhersage dieser Aktivität ausgehend vom Genotyp verbessern lassen sollte. Besonders für CYP2D6 ermöglicht das neue Korrelationsmodel präzisere Vorhersagen des Phänotyps als bisher veröffentlichte Modelle. Zusammengefasst leistet diese Studie durch die Entwicklung eines sicheren und einfachen Phänotypisierungsprozesses für CYP2D6 und CYP2C9 und durch die Bestimmung von Aktivitätskoeffizienten für alle einbezogenen CYP2D6 und CYP2C9 Allele und der damit verbundenen präziseren Vorhersage des Phänotyps ausgehend vom Genotyp einen wesentlichen Beitrag zum Forschungsfeld der Pharmakogenetik. / This study should contribute to the important field of pharmacogenetics by: firstly, establishing an easy and safe phenotyping method that combines the activity determination of all three previously mentioned CYPs (CYP2D6, CYP2C9, and CYP2C19) into one phenotyping cocktail and secondly, improving the knowledge about the predictive power of the genotype for the measured phenotype. It was indeed possible to develop a save, easy-to-use, fast and simultaneous phenotyping procedure for the important genetic polymorphic enzymes CYP2D6 and CYP2C9. To accomplish that, interaction studies with the chosen probe drugs dextromethorphan (DEX, CYP2D6), flurbiprofen (FLB, CYP2C9) and omeprazole (OME, CYP2C19) were conducted. It could be proven that DEX and FLB can be administered in combination, whereas OME alters the phenotyping results of CYP2C9. This is a new finding as in 2004 a phenotyping cocktail was published that used FLB and OME in combination. However, to our knowledge, no interaction tests were carried in that study. The new phenotyping procedure is not only verified by prior probe drug interaction studies, it also has other advantages over phenotyping cocktails found in literature. Firstly, save probe drugs are used in very small doses. This is possible due to the new sensitive LC-MS/MS methods that were evaluated. Secondly, the new phenotyping procedure is very fast and on-invasive. Urine has to be collected only for 2 h and the results also suggest that the time consuming glucuronide cleavage of the CYP2D6 dependent metabolite dextrorphan, usually carried out before CYP2D6 phenotyping, may be unnecessary. Most importantly, however, new insights into the phenotype prediction from genotype for CYP2C9 and CYP2D6 could be gained within this study. Nearly 300 phenotyped Caucasian subjects were also genotyped for the most important known variant alleles for CYP2D6, CYP2C9 and CYP2C19 using several established and newly developed genoptyping methods. Therefore, a direct correlation between phenotype and genotype could be conducted for CYP2D6 and CYP2C9. Employing linear modeling, it was possible to assign activity coefficients to each of the detected CYP2D6 and CYP2C9 alleles, thereby estimating their contribution to the resulting enzyme activity. This might facilitate the prediction of the CYP2D6 and CYP2C9 metabolic status of a subject knowing only its respective genotypes. Especially the new CYP2D6 genotype phenotype correlation model might allow for more precise phenotype prediction for the included variant alleles than was possible until now. Taken together, this study substantially contributes to the important research field of pharmacogenetics by (i) developing a save and easy-to-use phenotyping combination for CYP2D6 and CYP2C9, and (ii) by establishing activity coefficients for each of the detected CYP2D6 and CYP2C9 alleles, thereby allowing for a more precise prediction of the phenotype from genotype.

Secreted proteins play important roles in many cellular functions and molecular processes. Because secreted proteins potentially enter the blood stream, they can serve as valuable measures of health and disease useful for disease diagnosis and prognosis, therapeutic target identification, and patient stratification in personalized medicine. Consequently, significant interest exists in secreted protein analysis within complex biospecimens, particularly blood but significant bioanalytical challenges including the wide protein dynamic range >10 orders of magnitude remain. The cellular secretome therefore represents a viable alternative to direct biomarker discovery in biofluids. Finally, cellular systems are amenable to labeling for the production of intact stable isotope labeled (SIL) proteins that can be used as global internal standards for quantitative proteomics. In this dissertation, two secretome-focused studies were undertaken. The first study involving candidate biomarker discovery in radiation-induced autophagy utilized the p53-null and inducible H1299 non-small cell lung cancer (NSCLC) secretome. The study identified 364 secreted proteins that were mainly associated with exosomes (N=224) and chaperone activity (N=21). CHGB and SCG2 were identified as potential population-based biomarkers (for patient stratification) due to their consistent overexpression in p53-null H1299 cell secretomes compared to p53-wt cells before and after radiation. FAM3C, CANX, EIF5A, GPI, and TXNRD1 were identified as candidate biomarkers for patient prognosis following radiotherapy due to their differential expression only in response to radiation treatment. In the second study, a comprehensive glycoproteomics characterization of the SILAC-labeled HepG2 secretome was undertaken. 1635 SIL proteins, 492 of which were major plasma proteins including 192 cancer biomarkers were identified with high sequence coverage spanning six orders of magnitude. EDTA plasma spiked with the SIL secretomes yielded 63 proteins that were quantified with H/L ratios in all samples out of 1405 total proteins identified. Additionally, LC-MS/MS analysis of the Con A and WGA enriched 72h secretome:plasma sample afforded an opportunity to clearly distinguish between glycoproteins in plasma and the HepG2 secretome that share/differ in N-glycan structures. Collectively, the two studies reveal the suitability of the H1299 cancer cell secretome as an experimental model for biomarker studies and support the HepG2 secretome as a viable platform for producing SIL glycoproteins.

Resumo: Produtos cárneos que contenham propriedades organolépticas específicas e que atendam às exigências dos consumidores, são cada vez mais valorizados no mercado. Apesar da sua importância, as características de qualidade da carne em geral não são consideradas em programas de melhoramento genético animal, em razão do alto custo e dificuldade de mensuração. Marcadores moleculares identificados em análises genômicas, têm sido estudados em rebanhos Nelore. Trabalhos que visam o entendimento da expressão de genes e proteínas envolvidos na determinação de características relacionadas a gordura intramuscular da carne, são realizados com o intuito de auxiliar no entendimento da expressão dos genes relacionados a possíveis biomarcadores. Assim, inúmeros trabalhos que visam avaliar a expressão diferencial de genes podem ser encontrados na literatura, no entanto, devido a divergências entre o manejo e as raças estudadas, os resultados são discordantes, e a validação dos genes mais comumente encontrados, se faz necessário para que essas informações possam ser úteis em programas de melhoramento genético. Assim, este estudo teve como finalidade buscar na literatura genes diferencialmente expressos associados à característica de deposição de gordura intramuscular, avaliada por meio de duas técnicas, e validá-los em uma população de bovinos Nelore com fenótipo extremo para a característica gordura intramuscular por meio da técnica de PCR quantitativa em tempo real. Além disto, foi verificado se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Meat products with specific organoleptic properties and that attend the requirements of consumers, are increasingly valued in the market. Despite its importance, meat quality traits have not been widely used in animal breeding programs because of the high cost and difficulty of measurement. Molecular markers identified in genomic analyzes have been studied in Nellore herds. Studies aimed at understanding the expression of genes and proteins involved in the determination of characteristics such as intramuscular fat from meat are performed with the aim of helping to understand the expression of the genes related to possible biomarkers. Several studies that aim the expression of differentially expressed genes can be found in the literature, however, because of differences between management and breeds studied the results are discordant, and the validation of the common genes found is necessary for this information to be useful in breeding programs. Thus, this study aimed to search the literature for differentially expressed genes related to the marbling trait, by means of the two techniques for the evaluation of this trait and to validate them in a commercial herd of Nellore cattle, by quantitative real time PCR techniques. Besides that, this study will verify if the differentially expressed genes are being translated into differentially expressed proteins, using advanced mass spectrometry technique (LC-MS/MS). For this, 24 divergent samples were selected for each intramuscular ... (Complete abstract click electronic access below) / Mestre

In the last few years, the prevalence of legal party pills in New Zealand has risen dramatically. These pills contain new piperazine designer drugs, two of the more common being 1-benzylpiperazine (BZP) and m-trifluoromethylphenylpiperazine (TFMPP). This thesis describes an optimised LC-MS/MS method for the detection of BZP and TFMPP in whole blood, using an automated solid phase extraction (SPE) for sample clean-up. The method was validated on three different days using five replicate samples each day. The standard curve was linear from 7 - 7000 ng/mL for BZP and 10 - 10,000 ng/mL for TFMPP, with coefficients of variation (CV) below 10%, and accuracy greater than 90% for both drugs. The method was used to quantitate samples provided by the Medical Research Institute of New Zealand. Blood levels were used to show concentrations in the blood over time, and relate these to performance of subjects on a driving simulator. The study was stopped after 41% of the participants who received BZP and TFMPP had adverse reactions to the pills, including vomiting and migraines. The LC-MS/MS method was also used to detect and quantitate methamphetamine, amphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, morphine, codeine and 6-monoacetylmorphine in hair. The drugs were extracted from 20 mg of hair using hydrochloric acid in a water bath overnight, then purified using SPE. Validation on three days with five replicate samples gave coefficients of variation (CV) below 12% and acceptable accuracy for all drugs. The method was tested on three samples, previously reported by Environmental Science and Research (ESR) using gas chromatography mass spectrometry (GC-MS) giving results in good agreement. This thesis describes a sensitive, accurate, reproducible LC-MS/MS method easily adapted to analyse drugs of abuse in different biological matrices. It demonstrates the versatility of LC-MS/MS and its applications in forensic work.

La thèse a porté sur l'étude de peptides et d'oligonucléotides par fragmentation dans la source électrospray d'un Quattro II (Micromass). L'instrument a d'abord été évalué en mode positif avec des peptides thioester modèles puis en mode négatif avec des oligodésoxynucléotides bromés dont la fragmentation de la base modifiée permet sa localisation dans la séquence. La fragmentation de peptides acétal et diol (aldéhyde hydraté) conduit à un même ion final cyclique. L'hydratation de peptides aldéhyde a été étudiée en solution par RMN et en phase gazeuse par fragmentation dans la source. Les informations issues de ce travail ont permis une meilleure compréhension du mécanisme de la réaction d'oximation.

<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>

This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.

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. 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 tert-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.