Tesis: "LC-MS methods"

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Zhao, Ruoxia. "Investigation of Ribonucleic Acid Post-transcriptional Modifications by Optimized LC-MS/MS Methods". University of Cincinnati / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1623240214971428.

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2

Sharp, Julia Lynn. "New Statistical Methods for Analyzing Proteomics Data from Affinity Isolation LC-MS/MS Experiments". Thesis, Montana State University, 2007. http://etd.lib.montana.edu/etd/2007/sharp/SharpJ0807.pdf.

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The field of proteomics is exploding with statistical problems waiting to be explored. To obtain information on protein complexes, interactions between protein pairs is initially examined. This exploration is performed using `bait-prey' protein pull-down assays that use a protein affinity agent and an LC-MS/MS (liquid chromatography-tandem mass-spectrometry)-based protein identifcation method. An experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. The prey protein presence/absence pattern is assessed with a Likelihood Ratio Test (LRT) and simulated LRT p-values. Fisher's Exact Test and a conditional frequency distribution test using generating functions are also used to assess the prey protein observation pattern. Based on the p-value, each prey protein is assigned a category (Specific or Non-Specific) and appraised with respect to the goal and design of the experiment. The Bayes' Odds is calculated for each prey-bait pair in the `Specific' category to estimate the posterior probability that two proteins interact and compared to an approach used by Gilchrist et al. [23]. The method is illustrated using an experiment investigating protein complexes of Shewanella oneidensis MR-1 at the Proteomics Facility of Pacific Northwest National Laboratory (PNNL). The example analysis shows the results to be biologically sensible and more realistic than methods previously used to infer protein - protein associations. While inferring protein-protein associations is of great importance in proteomic studies, the quality of the data is of equal or greater importance. Protein-protein interactions may be inferred incorrectly or not at all depending on the quality of the data. Prior to this thesis, statistical quality control measures have not been incorporated into these experiments. The implementation of traditional Individual/Moving Range (IMR) charts and cumulative sum (cusum) quality control methods for use with pull-down experiment data is studied. These methodologies are illustrated using a standard protein mixture from PNNL. The joint application of IMR and cusum charts promises to provide researchers with information on changes in the mean and variability of the data resulting from control samples run through the mass spectrometer process.

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3

Li, Lan. "Development of Quantitative LC-MS/MS Methods for the Pharmacological Studies of Anti-Cancer Drugs". Cleveland State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1299117212.

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4

Nezami, Ranjbar Mohammad Rasoul. "Novel Preprocessing and Normalization Methods for Analysis of GC/LC-MS Data". Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73499.

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We introduce new methods for preprocessing and normalization of data acquired by gas/liquid chromatography coupled with mass spectrometry (GC/LC-MS). Normalization is desired prior to subsequent statistical analysis to adjust variabilities in ion intensities that are not caused by biological differences. There are different sources of experimental bias including variabilities in sample collection, sample storage, poor experimental design, noise, etc. Also, instrument variability in experiments involving a large number of runs leads to a significant drift in intensity measurements. We propose new normalization methods based on bootstrapping, Gaussian process regression, non-negative matrix factorization (NMF), and Bayesian hierarchical models. These methods model the bias by borrowing information across runs and features. Another novel aspect is utilizing scan-level data to improve the accuracy of quantification. We evaluated the performance of our method using simulated and experimental data. In comparison with several existing methods, the proposed methods yielded significant improvement. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the technologies widely used for qualitative and quantitative analysis of small molecules. In particular, GC coupled to single quadrupole MS can be utilized for targeted analysis by selected ion monitoring (SIM). However, to our knowledge, there are no software tools specifically designed for analysis of GS-SIM-MS data. We introduce SIMAT, a new R package for quantitative analysis of the levels of targeted analytes. SIMAT provides guidance in choosing fragments for a list of targets. This is accomplished through an optimization algorithm that has the capability to select the most appropriate fragments from overlapping peaks based on a pre-specified library of background analytes. The tool also allows visualization of the total ion chromatogram (TIC) of runs and extracted ion chromatogram (EIC) of analytes of interest. Moreover, retention index (RI) calibration can be performed and raw GC-SIM-MS data can be imported in netCDF or NIST mass spectral library (MSL) formats. We evaluated the performance of SIMAT using several experimental data sets. Our results demonstrate that SIMAT performs better than AMDIS and MetaboliteDetector in terms of finding the correct targets in the acquired GC-SIM-MS data and estimating their relative levels.
Ph. D.

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5

Minten, Johanna. "Development of methods for the analysis of polar compounds in environmental matrices using LC/UV and LC/MS /". Stockholm : Department of applied environmental science (ITM), Stockholm University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-29108.

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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2009.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Submitted. Härtill 4 uppsatser.

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Cai, Xiaohan. "¿¿¿¿¿¿Development of Bioanalytical Methods for Clinical Applications and Drug Screening". Cleveland State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1314982525.

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7

Abbas, Ioana. "Development of LC-MS/MS methods for the quantitative determination of hepcidin-25, a key regulator of iron metabolism". Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19358.

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Hepcidin-25, ein 2000 entdecktes Peptidhormon, das eine Schlüsselrolle im Eisenstoffwechsel spielt, hat das Verständnis von Eisenerkrankungen revolutioniert. In dieser Studie wurde LC gekoppelt mit einem Triple-Quadrupol MS in einer schnellen und robusten Methode zur Quantifizierung von Hepcidin-25 in menschlichem Serum verwendet, welche letztlich in Routine-Laboratorien genutzt werden soll. Zu diesem Zweck wurden zwei Probenvorbereitungsstrategien und zwei komplementäre LC Bedingungen untersucht, wobei eine saure mobile Phase (0,1% TFA) mit einem neuartigen Ansatz unter der Verwendung einer basischen mobilen Phase (0,1% NH3) verglichen wurde. In einem laborinternen Vergleich beider LC-MS/MS Methoden wurde Hepcidin-25 in humanen Proben unter Verwendung der gleichen Kalibrierstandards quantifiziert und eine sehr gute Korrelation der Ergebnisse ermittelt. Hierbei wurde die Analysestrategie mit saurer mobiler Phase als hochsensitiv (LOQ von 0,5 μg/L) und präzise (CV <15%) befunden und als Kandidat einer Referenzmethoden für die Hepcidin-25 Quantifizierung in realen Proben empfohlen. Einer der neuartigen Aspekte der Methodik war die Verwendung von Amino- und Fluor-silanisierten Autosampler-Fläschchen, um die Adsorption des 25 Reste umfassenden Peptids anOberflächen zu reduzieren. Darüber hinaus wurde diese LC-MS/MS-Methode in einer internationalen Ringversuchsstudie eingesetzt, bei der ein sekundäres Referenzmaterial als Kalibrierstandard verwendet wurde, und gemäß des International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR) als optimal bewertet. In dieser Arbeit wurde die Bildung von Hepcidin-Komplexen mit Kupfer(II) untersucht. Die erste Umkehrphasen-chromatographische Trennung von Hepcidin-25/Cu(II) und Hepcidin-25 (Kupfer "frei") wurde unter Verwendung von mobilen Phasen mit 0,1% NH3 erreicht. LC-MS/MS und FTICR-MS wurden für die Charakterisierung der gebildeten Hepcidin-25-Cu(II)-Spezies bei pH-Werten von 11 bzw. 7,4 verwendet.
Hepcidin-25, a key iron-regulatory peptide hormone discovered in 2000, has revolutionized the understanding of iron-related pathology. This study applied LC-MS/MS, using the triple quadrupole mass spectrometer, in a rapid and robust analytical strategy for the quantification of hepcidin-25 in human serum, to be implemented in routine laboratories. For this purpose, two sample preparation strategies and two complementary LC conditions were investigated, where the use of acidic mobile phases (0.1% TFA) was compared with a novel approach involving solvents at high pH (0.1% NH3). The application of these LC-MS/MS methods to human samples in an intra-laboratory comparison, using the same hepcidin-25 calibrators, yielded a very good correlation of the results. The LC-MS/MS employing trifluoroacetic acid-based mobile phases was selected as a highly sensitive (LOQ of 0.5 µg/L) and precise (CV<15%) method and was recommended as a reference method candidate for hepcidin-25 quantification in real samples. One of the novel aspects of the methodology was the use of amino- and fluoro-silanized autosampler vials to reduce the interaction of the 25-residue peptide to laboratory glassware surfaces. Moreover, this LC-MS/MS method was used for an international round robin study, applying a secondary reference material as a calibrator and its performance was found to be in the optimal range as defined by the International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR). In this work, the formation of hepcidin-25 complexes with copper(II) was investigated. The first reversed-phase chromatographic separation of hepcidin-25/Cu2+ and hepcidin-25 (copper “free”) was achieved by applying mobile phases containing 0.1% NH3. LC-MS/MS and FTICR-MS were applied for the characterization of the formed hepcidin-25-Cu(II) species at pH values of 11 and 7.4 respectively. A new species corresponding to hepcidin-25 complexed with two copper ions was identified at high pH.

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Barclay, Victoria K. H. "Development of LC-MS/MS Methods for the Analysis of Chiral and Achiral Pharmaceuticals and Metabolites in Aqueous Environmental Matrices". Doctoral thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171550.

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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.

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Wabuyele, Simuli Lindah. "DEVELOPMENT OF LC-MS/MS METHODS FOR QUANTITATIVE ANALYSIS OF PLANT-DERIVED ANTICANCER AGENT AND SYNTHETIC ESTROGEN IN COMPLEX MATRICES". Cleveland State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1400262843.

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10

Fujisawa, Alma. "Development of chemical labeling methods for organelle molecule analysis". Kyoto University, 2019. http://hdl.handle.net/2433/243315.

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11

Wilson, Michael Andrew. "Analysis of complex distributions of bacteriochlorophylls : development and application of HPLC and LC-MS methods". Thesis, University of York, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422530.

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12

Strömqvist, Malin. "Development of quantitative methods for the determination of vemurafenib and its metabolites in human plasma". Thesis, Linköpings universitet, Kemi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-110076.

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Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe. LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®. A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria. The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.

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13

Upite, Ruta, Susanna Wärmegård, Casper Tiger, Nordén Anna Ivert, Temis Martinez y Viktor Umenius. "Immunoassays or LC-MS/MS? : A Comparison Revealing the Properties of Modern Methods for Insulin, Pro-insulin, C-peptide and Glucagon Quantification". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-384690.

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The purpose of this report is to compare seven different methods for biomarker detection and quantification based on previously published papers. The methods investigated are ELISA, LC-MS/MS, UPLC-MS/MS, LC-IM/MS, IA-LC-MS/MS, MSIA-HR/AM, HTRF and AlphaLISA ® . The focus lies on biomarkers relevant for diabetes, obesity and cardiovascular diseases.Namely insulin, proinsulin, glucagon and C-peptide. Particular significance is assigned to the comparison of the currently widest used method, ELISA, with various types of LC-MS/MS. The report concludes ELISA being superior to LC-MS/MS methods in terms of recovery and precision, while LC-MS/MS is superior in accuracy, multiplexing, specificity, throughput and sample cost. This suggests that different types of LC-MS/MS has the potential to gain momentum in the field of biomarker quantification if they become more available.

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14

Ly, Tuan Kiet. "Development of analytical methods of multi-pesticide residues for controlling the tea quality, from tea plantation to consumer". Thesis, Toulouse, INPT, 2020. http://www.theses.fr/2020INPT0059.

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Le thé est la deuxième boisson la plus consommée au monde, dépassé uniquement par l'eau, en raison de ses bienfaits pour la santé. Cependant, en raison des pratiques de culture en monoculture, l'utilisation de pesticides pendant la culture du thé est courante. Au fil du temps, le nombre de pesticides utilisés a augmenté et, pour protéger la santé des consommateurs, de nombreux pays et régions ont établi des limites maximales de résidus de pesticides pour une variété d'aliments et de boissons, y compris le thé. Par exemple, l'Union européenne (UE) a fixé les limites maximales de résidus (LMR) pour plus de 480 pesticides et leurs métabolites dans les produits à base de thé. Par conséquent, le développement de méthodes analytiques pour les résidus multi-pesticides dans le thé est un défi, car le thé est un produit complexe avec de nombreux composés qui peuvent interférer avec les résultats, tels que les polyphénols, les pigments et la caféine. Le but de cette thèse est de développer des méthodes robustes et robustes avec une sensibilité, une exactitude et une précision élevées pour répondre aux réglementations de l'UE pour la détermination simultanée de 400 résidus de pesticides dans les produits de thé en utilisant des chromatographies liquides et gazeuses ultra performantes couplées à la spectroscopie de masse en tandem (UPLC- MS/MS et GC-MS/MS, respectivement). La première partie de la thèse portait sur l'élimination des effets de matrice dans les feuilles de thé vertes en combinant l'extraction QuEChERS (rapide, facile, bon marché, efficace, robuste et sûre) et le nettoyage en mode mixte SPE (extraction en phase solide). Une cartouche SPE C18 couplée à SPE GCB / PSA s'est avérée être la méthode de nettoyage la plus efficace et a permis de quantifier 225 résidus de pesticides, sur la base des courbes d'étalonnage des solvants (154 résidus utilisant UPLC-MS/MS et 71 résidus utilisant GC-MS/MS). Les méthodes analytiques ont été entièrement validées conformément au SANTE/11945/2015 (UE). Les LOQ pour la plupart des pesticides (386/400 ou 96,5%) étaient inférieures à 10 μg / kg, c'est-à-dire inférieures à la LMR de l'UE (5-70 mg / kg). Dans la deuxième partie, les effets matriciels de 400 résidus de pesticides ont été étudiés et améliorés pour l'analyse de différents types de thés (blancs, verts, oolongs et noirs). Les résultats ont montré que la combinaison de l'extraction QuEChERS et du nettoyage SPE en mode mixte et après la réduction du volume d'injection s'est avérée être la procédure la plus efficace pour surmonter les effets de matrice. Plus de 190 pesticides (> 95% des 200) ont eu l'effet matriciel dans une fourchette de ± 20% pour UPLC-MS/MS. Par conséquent, ils peuvent être quantifiés à l'aide de courbes d'étalonnage de solvant. D'un autre côté, des courbes d'étalonnage adaptées à la matrice doivent être utilisées pour surmonter les effets de matrice pour GC-MS/MS. De plus, nous avons reconnu que les effets de matrice dans GC-MS/MS n'étaient pas seulement une amélioration du signal mais aussi une suppression. Enfin, dans la troisième partie de ces travaux, la méthode établie a été appliquée avec succès à la détermination des résidus multi-pesticides dans 106 échantillons de thé. Au total, 26 échantillons de thé (24,5%) contenaient au moins une violation de pesticides, avec 43 violations de résidus de pesticides. Les pesticides les plus fréquemment détectés étaient les néonicotinoïdes, les pyréthrinoïdes synthétiques et les fongicides triazolés. En termes d'origine dans cette étude, Taïwan avait les échantillons les plus contaminés par les pesticides avec 83,3%, suivi par la Chine (73,7%), le Vietnam (64,7%) et l'Inde (Darjeeling) (55,0%). Les résultats ont montré que les échantillons dépassant les réglementations de l'UE en matière de LMR étaient toujours élevés à 24,5%. Par conséquent, les évaluations des multiples résidus de pesticides dans le thé doivent être poursuivies
Tea is the second most-consumed beverage in the world, surpassed only by water, due to its health benefits. However, because of monoculture cultivation practices, the use of pesticides during tea cultivation is common. Over time, the number of pesticides used has increased, and, to protect consumers' health, many countries and regions have established maximum residue limits of pesticides for a variety of foods and beverages, including tea. For instance, the European Union (EU) has set the maximum residue limits (MRLs) for more than 480 pesticides and their metabolites in tea products. Therefore, the development of analytical methods for multi-pesticide residues in tea is a challenge, because tea is a complex commodity with many compounds that can interfere with results, such as polyphenols, pigments, and caffeine. The aim of this thesis is to develop rugged and robust methods with high sensitivity, accuracy, and precision to meet the EU regulations for simultaneous determination of 400 pesticide residues in tea products using ultra performance liquid and gas chromatographies coupled to tandem mass spectroscopy (UPLCMS/ MS and GC-MS/MS, respectively). The first part of thesis focused on elimination of matrix effects in green tealeaves by combining QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction and mixed-mode SPE (solid phase extraction) clean-up. A C18 SPE cartridge paired with SPE GCB/PSA proved to be the most effective clean-up method and enabled 225 pesticide residues to be quantified, based on solvent calibration curves (154 residues using UPLCMS/ MS and 71 residues using GC-MS/MS). The analytical methods were validated fully in accordance with the SANTE/11945/2015 (EU). LOQs for most pesticides (386/400 or 96.5%) were below 10 μg/kg, i.e., less than the EU MRL (5-70 mg/kg). In the second part, matrix effects for 400 pesticide residues were investigated and improved for the analysis of different types of teas (white, green, oolong and black ones). Results showed that combining QuEChERS extraction and mixed-mode SPE clean-up, and following the reduction of the injection volume were found to be the most effective procedure to overcome matrix effects. More than 190 pesticides (> 95% of the 200 ones) had the matrix effect within the range of ± 20% for UPLC-MS/MS. Therefore, they can be quantified using solvent calibration curves. On the other hand, matrix-matched calibration curves should be used to overcome matrix effects for GC-MS/MS. Moreover, we recognized that matrix effects in GC-MS/MS were not only signal enhancement but also suppression. Finally, in the third part of this work, the established method was successfully applied to the determination of multi-pesticide residues in 106 tea samples. In total, 26 tea samples (24.5%) were containing at least one pesticide violation, with 43 pesticide residue violations. The most frequently detected pesticides were neonicotinoids, synthetic pyrethroids, and triazole fungicides. In terms of origin in this study, Taiwan had the most pesticide-contaminated samples with 83.3%, following by China (73.7%), Vietnam (64.7%), and India (Darjeeling) (55.0%). The results showed that samples exceeding EU MRLs regulations were still high with 24.5%. Therefore, assessments of multipesticide residues in tea need to be continued

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Wiegandt, Alena [Verfasser] y Bernd [Akademischer Betreuer] Meyer. "New Methods for Characterization of N-type Glycosylation of Proteins by Integration of LC-MS/MS and NMR / Alena Wiegandt ; Betreuer: Bernd Meyer". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1209272407/34.

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Bärenfänger, Melissa [Verfasser] y Bernd [Akademischer Betreuer] Meyer. "New Methods for the Characterization of Human Glycoproteins by LC-MS/MStechniques / Melissa Bärenfänger ; Betreuer: Bernd Meyer". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1205070834/34.

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17

Huang, Qianyang. "Development and Validation of UPLC/MS/MS Methods for Quantification of Gangliosides in the Clinical Study of Ganglioside GM3 Synthase Deficiency". Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1472042152.

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Lehto, T. (Tiina). "Evaluation of new laboratory methods for routine use". Doctoral thesis, Oulun yliopisto, 2016. http://urn.fi/urn:isbn:9789526210759.

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Abstract Laboratory medicine is under constant pressure from changes in the operating environment. Organisational changes and tendering processes have led to a trend towards shorter turn-around times and more cost-effective choices. Analysis tools that were previously only available at research laboratories, such as the mass spectrometer and polymerace chain reaction (PCR), have now made their way to university hospital laboratories and even mid-sized laboratories. Organisational changes have increased the need to monitor the pre-analytical steps. The specimen can be drawn from the patient in a satellite laboratory, which may be located several hours from the central laboratory. The increased transportation times may change the analytical properties of the specimens, which is why the stability of different analytes should be investigated thoroughly in different temperatures. It should be born in mind that doctors are treating the patients based on the results they receive from the laboratory. To avoid possible malpractice, the analytical properties should remain reliable. Traditionally, some analyses have been carried out manually, which is known to be time-consuming and carries the possibility of wide intra-observatory mistakes. For that reason, it would be reasonable to perform some manual analyses, such as body fluid analysis, in an automated manner. Automating the manual steps taken in the laboratory would release labour for other tasks and may increase the cost-effectiveness of the work. Organisational changes have redirected the needs of a clinical laboratory towards automated options instead of manual ones and finding more economically-based alternatives to replace or complement traditional methods
Tiivistelmä Laboratoriolääketiede on jatkuvan muutospaineen alla. Organisaatiomuutokset ja kilpailutus ovat saaneet aikaan sen, että laboratorioiden analytiikkatarjonnan tulee olla kilpailukykyistä niin hinnan kuin tulosten vastausnopeuden suhteen. Aikaisemmin pelkästään tutkimuskäytössä olleet menetelmät, kuten PCR ja massaspektrometri, ovat jalkautuneet jo keskussairaalatasoiseen tutkimusvalikoimaan. Organisaatiomuutokset ovat saaneet aikaan myös sen, että näytteet voidaan ottaa potilaasta alueellisissa toimipisteissä ja kuljettaa päivän aikana keskuslaboratorioon analysoitavaksi. Kuljetusmatkat ja -ajat saattavat olla hyvinkin pitkiä. Tämän johdosta on erittäin tärkeää selvittää näytteiden säilyvyys niin, että tulokset pysyvät luotettavina eikä potilaan hoito kärsi. Perinteisesti osa tutkimuksista, kuten punktionesteen solut, on tehty käsin mikroskopoimalla, jonka tiedetään olevan aikaa vievää ja näin ollen myös kallista analysointia. Kyseisen tutkimuksen siirtäminen analysaattoreille tehtäväksi voi tuoda laboratoriolle taloudellisen säästön lisäksi työvoiman vapautumista manuaalisesti suoritettavalta mikroskopoinnilta. Muutospaineet laboratoriotoiminnoissa ovat saaneet aikaan tarpeen automatisaation lisääntymiselle ja taloudellisempien vaihtoehtojen löytämiselle perinteisten menetelmien rinnalle tai niiden sijaan

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Zeng, Chun. "The Role of RNASE L in Type I Diabetes and Development of Quantitative LC-MS/MS Methods for the Pharmacological Studies of Anti-Cancer Drug Candidates". Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1473178407.

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20

Jakob-Rodamer, Verena [Verfasser], Fritz [Gutachter] Sörgel, Ulrike [Gutachter] Holzgrabe y Petra [Gutachter] Högger. "Development and validation of LC-MS/MS methods to determine PK/PD parameters of anti-infectives / Verena Jakob-Rodamer. Gutachter: Fritz Sörgel ; Ulrike Holzgrabe ; Petra Högger". Würzburg : Universität Würzburg, 2015. http://d-nb.info/1112040285/34.

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Archibald, Timothy L., Derek Edward Murrell y Stacy D. Brown. "Chromatographic Methods in Hiv Medicine: Application to Therapeutic Drug Monitoring". Digital Commons @ East Tennessee State University, 2018. https://doi.org/10.1002/bmc.4170.

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HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid-liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18 ). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

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Rodamer, Michael [Verfasser] y Ulrike [Akademischer Betreuer] Holzgrabe. "Development of practice-oriented LC-MS, MS methods for the determination of important drugs and their application for building PK, PD concepts / Michael Rodamer. Betreuer: Ulrike Holzgrabe". Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/102252268X/34.

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23

NOBILE, MARIA. "DEVELOPMENT AND VALIDATION OF METHODS FOR THE DETECTION OF RESIDUES IN UNCONVENTIONAL AND INNOVATIVE MATRICES THROUGH LC-MS/MS ANALYSES FOR SAFETY OF FOOD OF ANIMAL ORIGIN". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/547683.

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Successful animal growth depends on a combination of many factors related to health, management and nutrition. The use of veterinary drugs in food-producing animals for therapeutic purposes is regulated (corticosteroids, antibiotics) or banned (anabolic steroids) in the European Union; however, their use as growth promoters cannot be excluded. Moreover, the eventual presence of residues in food constitutes a fraud and a health issue for the consumers. For these reasons the need to find new accumulation matrices and new sensitive, specific and robust methods that are able to reveal the presence of drug residues is essential, based on the fact that there is a low percentage of non-conformity in the final reports of the National Residues Plan in recent years, although the threat of a disproportionate use of these substances is increasingly on the rise. In the light of these facts, there is the need to implement the framework of controls aimed to food safety, due to the inefficiency of tools for the study of these substances. Often, the use of conventional matrices, such as urine, liver or muscle, recommended for the official controls of illegal treatment are not completely satisfactory due to the fast elimination rate of the compounds or to the difficulties arising from the compounds characterised also by a pseudoendogenous nature. The debate about the presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or illicit treatment, is currently ongoing within the European Union. These compounds have been appropriately defined “grey-zone substances”, for their double origin. The simple detection of some steroids in urine is currently considered to provide insufficient evidence of illicit treatment. Parameters such as cut-off levels, the presence of metabolites, or both, must be accounted for. As regards antibiotics, the overuse, over the last decades, as growth promoters in food producing animal have caused favorable condition about the threat of bacterial resistance. The antibiotics can directly affect the consumer in the form of residues from the food chain, or by accumulation in the environment via the application of manure to land as organic fertiliser, via sludge storage or by direct contamination of illicitly additivated water and feed. The main challenge is to monitor contemporally different antibiotic classes, in different steps of the food chain, trying to control this phenomenon. On the other hand, food contamination by new environmental contaminants should not be neglected. In particular, perfluoroalkyl substances (PFASs) have recently aroused great scientific interest and concern for public health, due to the fact they have been found in appreciable concentrations in human serum. On the basis of EFSA requestes and of analytical problems associated with their determination many studies are recommended to monitor their presence, building a database on PFASs in food, evaluate the contamination levels of the individual compound and finally draw up a reliable risk assesstment of European population. This work was born with the aim to detect residues of the most commonly used drugs in broad sense, and then extended over time, also following requests from public and private entities, based on realistic situations of risk. Therefore, based on the mentionated issues, the development, optimisation and validation of multiresidual methods and the direct application on real unconventional matrices allowed us to have a greater amount of information in terms of number, frequency, and concentration of different classes of veterinary drugs than in conventional matrices. We confirmed the presence of pseudoendogenous compounds and their precursors in the unconventional matrix bile, for example. The study of the unconventional matrices, e.g. bovine teeth, has also allowed us to detect esterified forms of some drugs, discriminating them from the active free forms that could have a double, exogenous and endogenous, origin. Finally, this work demonstrates the utility of an eventual introduction, through the food of animal origin chain, of several monitoring points of different types of residues, consisting of non-edible matrix analyses that are not destructive of the product intended for the consumer. On the other hand, the sensitivity and good performance of the developed LC-HRMS methods for the emerging PFASs, could help further studies and also EFSA to increase the number of quantifiable data useful to extend a risk assessment in its final reports.

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Van, Huyssteen Este. "Efficacy enhancement of the antimalarial drugs, mefloquine and artesunate, with PheroidTM technology / E. van Huyssteen". Thesis, North-West University, 2010. http://hdl.handle.net/10394/5050.

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Malaria is currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

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25

Desai, Meera Jay. "Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies". Ames, Iowa : Oak Ridge, Tenn. : Ames Laboratory ; distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy, 2004. http://www.osti.gov/servlets/purl/837266-GaBf1y/webviewable/.

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Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); 19 Dec 2004.
Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2134" Meera Jay Desai. US Department of Energy 12/19/2004. Report is also available in paper and microfiche from NTIS.

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26

Abbas, Ioana [Verfasser], Michael [Gutachter] Weller, Maria [Gutachter] Montes-Bayón y Michael [Gutachter] Linscheid. "Development of LC-MS/MS methods for the quantitative determination of hepcidin-25, a key regulator of iron metabolism / Ioana Abbas ; Gutachter: Michael Weller, Maria Montes-Bayón, Michael Linscheid". Berlin : Humboldt-Universität zu Berlin, 2018. http://d-nb.info/1185579397/34.

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27

Clarke, Rosemary Emily Jane. "The development and application of liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods for the quantification of the gut hormones peptide YY, glucagon-like peptide-1 and oxyntomodulin". Thesis, Imperial College London, 2018. http://hdl.handle.net/10044/1/59038.

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Our current understanding of gut hormones is limited by our inability to measure their concentration with sufficient specificity and sensitivity. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has already been applied to glucagon quantification and offers a potential solution. This PhD aimed to develop LC-MS/MS methods for the quantification of five peptide gut hormones: glucagon-like peptide-1 (GLP-1) 7-36 amide, GLP-1 9-36 amide, oxyntomodulin, peptide YY (PYY) 1-36 and PYY 3-36. The optimised extraction of the gut hormones from plasma comprised protein precipitation, followed by solid phase extraction using the Oasis MAX μElution plate. Sample extraction was customised to reduce protein and lipid interferents, with recoveries of 21-32%. Extraction was into a surrogate matrix of 20μg/mL bovine serum albumin, 20% acetic acid and 10% methanol developed to minimise non-specific adsorption. Stable-isotopically labelled internal standards for each gut hormone were used. The ultra performance liquid chromatography method on a Waters Peptide BEH C18 130Å column demonstrated good separation of the gut hormones from each other. The mobile phases were water (A), and acetonitrile (B), both with 0.1% formic acid. The flow rate was 0.35mL/min and the elution gradient 80-65% A over 9 minutes. A Waters Xevo TQ-S triple quadrupole mass spectrometer was used in multiple reaction monitoring mode. Ionisation was in positive mode using atmospheric pressure electrospray ionisation. Specific precursor and product ions were measured for each gut hormone and internal standard. Spiked plasma samples for calibration line standards and quality control samples were measured. The method was imprecise with coefficients of variation >15%. The method could not be validated. Non-specific adsorption of the peptide gut hormones occurred despite optimisation of container type, temperature, solvent and sample handling. Specificity was improved in comparison to immunoassay. Future work will concentrate on PYY species and aim to improve the sample extraction recovery and precision.

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28

Smit, Michiel Johannes. "Development and validation of selective and sensitive LC-MS/MS Methods for determination of para-aminosalicyclic acid and cycloserine/terizidone applicable to clinical studies for the treatment of tuberculosis". Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29814.

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A method was validated for the quantification of para-aminosalicylic acid (PAS) in human plasma. The technique consisted of a protein precipitation extraction, followed by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) detection. Rilmenidine was used as the internal standard (ISTD). Analyte mean extraction yields determined were ~100.3% (CV % = 3.3). The extraction procedure was followed by liquid chromatographic separation using a Phenomenex Synergi Hydro-RP (150 x 2.0 mm, 4µm) analytical column. An isocratic mobile phase containing methanol, water and formic acid (40:59.8:0.2, v/v/v) was used at a flow-rate of 300 µl per minute. The retention times for PAS and rilmenidine were, ~2.4 and ~1.6 minutes, respectively. An AB Sciex API 3000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 154.1 and m/z 181.2 to the product ions m/z 80.2 and m/z 95.2 for PAS and the ISTD, respectively. Electro Spray Ionisation (ESI) was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for PAS over the range 0.391 – 100 µg/ml, based on peak area ratios. A 1:1 and 1:4 dilution of the QC Dilution sample showed that concentrations of up to 160 µg/ml of PAS in plasma could be analysed reliably when diluted into the calibration range. Endogenous matrix components were found to have an insignificant effect on the reproducibility of the method, when human plasma originating from eight different sources were analysed. PAS was found to be stable in human plasma for 21 months kept at ~-80°C, for up to 21 hours at room temperature and when subjected to 3 freeze-thaw cycles. Stock solutions of PAS in methanol were stable for 2 days when stored at ~80°C and for 24 hours when stored at room temperature, ~4°C and ~-20°C. Plasma extracts of the analyte/ISTD ratio were shown to be stable on instrument over a period of ~55 hours. Reinjection reproducibility experiments indicated that an assay batch may be re-injected within 58 hours. Quantification of PAS in plasma was not significantly affected by the presence of haemolysed blood (2%) in plasma and when Lithium Heparin was used as anti-coagulant instead of K3EDTA. The best marker for terizidone pharmacokinetics is the analysis of cycloserine, a small polar drug with limited potential for absorbing UV that makes it difficult to analyse. A method was validated for the quantification of cycloserine in human plasma, and consisted of a protein precipitation extraction and derivatization, followed by high performance liquid chromatography with MS/MS detection. No ISTD was used as no suitable match could be found. The mean extraction yield determined was ~77% (CV% = 10.7). The extraction procedure was followed by liquid chromatographic separation using a Gemini NX C18 (50 x 2.0 mm, 5µ) analytical column. An isocratic mobile phase containing acetonitrile, water and formic acid (30:69.9:0.1, v/v/v) was used at a flow-rate of 300 µl per minute. The retention time for cycloserine was ~ 1.5 minutes. An AB Sciex API 3000 mass spectrometer at unit resolution in the MRM mode was used to monitor the transition of the protonated precursor ion m/z 335.9 to the product ion m/z 157.2 for cycloserine. ESI was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for cycloserine over the range 0.313 – 40.0 µg/ml, based on peak areas. A 1:4 dilution of the QC Dilution sample showed that concentrations of up to 64.0 µg/ml of cycloserine in plasma could be analysed reliably when diluted into the calibration range and no carry over peaks were observed. Endogenous matrix components were found to have no effect on the reproducibility of the method when human plasma originating from six different sources was analysed. Cycloserine was found to be stable in human plasma for up to 18 hours at room temperature, and when subjected to 3 freeze-thaw cycles. Stock solutions of cycloserine in water and methanol were stable for 10 days when stored at ~ -80°C and for 18 hours when stored at room temperature, ~ 4°C and ~ -20°C. Long term stability in plasma has been proven for 17 months at -80°C. Plasma extracts of the analyte were shown to be stable on instrument over a period of ~ 29 hours. Reinjection reproducibility experiments indicate that an assay batch may be re-injected within 29 hours. Cycloserine is stable in whole blood (on ice) for up to 30 minutes. Both validated methods presented performed well on clinical samples generated from a multi drug resistant TB (MDR-TB) research study in children dosed with PAS and terizidone.

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Maria, Aoun. "Development of Analytical methods for the evaluation of the impact of phosphate fertilizer industry on marine environment". Thesis, Pau, 2014. http://www.theses.fr/2014PAUU3033.

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Développement de méthodes analytiques pour l’évaluation de l'impact de l'industrie de fertilisants chimiques sur le milieu marin
Development of Analytical methods for the evaluation of the impact of phosphate fertilizer industry on marine environment

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The, Matthew. "Statistical and machine learning methods to analyze large-scale mass spectrometry data". Licentiate thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-185149.

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As in many other fields, biology is faced with enormous amounts ofdata that contains valuable information that is yet to be extracted. The field of proteomics, the study of proteins, has the luxury of having large repositories containing data from tandem mass-spectrometry experiments, readily accessible for everyone who is interested. At the same time, there is still a lot to discover about proteins as the main actors in cell processes and cell signaling. In this thesis, we explore several methods to extract more information from the available data using methods from statistics and machine learning. In particular, we introduce MaRaCluster, a new method for clustering mass spectra on large-scale datasets. This method uses statistical methods to assess similarity between mass spectra, followed by the conservative complete-linkage clustering algorithm.The combination of these two resulted in up to 40% more peptide identifications on its consensus spectra compared to the state of the art method. Second, we attempt to clarify and promote protein-level false discovery rates (FDRs). Frequently, studies fail to report protein-level FDRs even though the proteins are actually the entities of interest. We provided a framework in which to discuss protein-level FDRs in a systematic manner to open up the discussion and take away potential hesitance. We also benchmarked some scalable protein inference methods and included the best one in the Percolator package. Furthermore, we added functionality to the Percolator package to accommodate the analysis of studies in which many runs are aggregated. This reduced the run time for a recent study regarding a draft human proteome from almost a full day to just 10 minutes on a commodity computer, resulting in a list of proteins together with their corresponding protein-level FDRs.

QC 20160412

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31

Westberg, Emelie. "Biomarkers of internal exposure/dose : Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates". Doctoral thesis, Stockholms universitet, Institutionen för material- och miljökemi (MMK), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-110490.

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This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

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Rapholo, Akanyang Annah Faithful. "Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry". Diss., University of Pretoria, 2017. http://hdl.handle.net/2263/63038.

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This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation.
Dissertation (MSc)--University of Pretoria, 2017.
Pharmacology
MSc
Unrestricted

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33

GOSETTI, FABIO. "Methods of analysis and control in food chemistry by chromatographic techniques with UV-Vis and mass spectrometry detections". Doctoral thesis, Università degli Studi del Piemonte Orientale, 2005. http://hdl.handle.net/10281/255650.

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Investigation in food science and technology, both by the food industry and governmental agencies, is based on the evaluation of food composition and characteristics. All food products require analysis as part of a quality management program that involves the development, the production processes and the control of the commercial product. The chemical composition and physical properties of food are necessary to evaluate the nutritive value, the functional characteristics and the acceptability of the food product. The nature of the sample and the specific use of the analysis dictate the choice of the analytical methods. Speed, precision, accuracy and ruggedness are often key factors in this choice. In any case the validation of the method for the specific food matrix is necessary to ensure the reliability of the method. Moreover, the success of any analytical method relies on the proper sampling selection and preparation of the food sample, on the careful execution of the analysis, and on the appropriate treatment and interpretation of the data. Methods of analysis developed and endorsed by nonprofit scientific organizations allow for standardized comparisons of the results between different laboratories, and for the evaluation of standard procedures. The official methods for the analysis of foods must ensure to meet the legal requirements established by governmental agencies. Food analysis can be defined as the discipline dealing with the development, application and comparison of the analytical procedures for characterizing the properties of foods and their constituents.

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Höfig, Carolin. "Establishment, validation and application of immunological and LC-MS/MS-based detection methods to study the role of human aromatic L-amino acid decarboxylase as an enzyme potentially involved in thyronamine biosynthesis". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16645.

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Thyronamine (TAM) sind eine neue Molekülklasse, die endokrinologische und metabolische Prozesse miteinander vereinen. Der biologisch aktive Metabolit 3-Iod-L-Thyronamin (3-T1AM) wird durch eine kombinierte Deiodierung und Decarboxylierung von Schilddrüsenhormonen (TH) gebildet. Existierende Methoden zum Nachweis und zur Quantifizierung von 3-T1AM im menschlichen Serum sind immer noch umstritten. Auch die an der Biosynthese vermutlich beteiligte TH-Decarboxylase konnte noch nicht identifiziert werden. Für die Identifizierung und Quantifizierung von TH und TAM Profilen wurde die Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS) verwendet. In der bisherigen präanalytischen Aufarbeitung liefern weder Flüssig-Flüssig- noch Festphasenextraktionen reproduzierbare Ergebnisse des 3-T1AM-Gehalts im Serum. Mit der Entwicklung eines spezifischen Extraktionsverfahrens und nachfolgender Detektion mittels LC-MS/MS gelang der gleichzeitige Nachweis der häufigsten TH im humanen Serum. Parallel dazu wurden monoklonale Antikörper gegen 3-T1AM entwickelt, auf deren Basis ein quantitativer 3-T1AM Chemilumineszenz-Immunoassay entstand. Ergebnisse aus klinischen Kollektiven zeigen, dass 3-T1AM im Serum im nM Konzentrationsbereich vorkommt und dass 3-T1AM bei Patienten außerhalb der Schilddrüse produziert wird. Viele Forscher gehen davon aus, dass die aromatische L-Aminosäure Decarboxylase (AADC) die Synthese von TAM über Decarboxylierung von TH katalysiert. Diese Hypothese wurde durch Inkubation von rekombinanter humaner AADC mit TH getestet. In keinem der Experimente konnte AADC die Decarboxylierung von TH katalysieren. Zusammenfassend ist die Bestimmung von 3-T1AM im Serum mittels LC-MS/MS aufgrund der nicht reproduzierbaren präanalytischen Probenaufbereitung problematisch. In dieser Arbeit wird der erste MAb-basierte 3-T1AM assay vorgestellt, der 3-T1AM zuverlässig in humanem Serum quantifiziert. Die AADC ist wahrscheinlich nicht an der Biosynthese von TAM beteiligt.
Thyronamines (TAM) are a new class of molecules linking endocrinology and metabolism. Combined deiodination and decarboxylation of thyroid hormones (TH) generates a biologically active ‘cooling’ metabolite, 3-iodo-L-thyronamine (3-T1AM).. It remains controversial, which methods are able or not to reliably detect 3-T1AM in human serum, and the presumed TH decarboxylase is still elusive. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the simultane-ous identification and quantification of TH and TAM profiles in biological samples. Several preanalytical methods were tested for complete extraction of 3-T1AM in human serum. Thus far, neither liquid-liquid nor solid-phase extraction methods allowed reproducible extraction of 3-T1AM from human serum samples in the preanalytical sample workup. Nevertheless, a rapid and sensitive extraction procedure was developed for detection of the major TH by LC-MS/MS in a single human serum sample. In parallel, monoclonal antibodies (MAb) targeting 3-T1AM were developed and characterized, and a highly specific quantitative 3-T1AM MAb-based chemiluminescence immunoassay was developed. Studies in clinical cohorts provide evidence that 3-T1AM is present in human serum in the nM concentration range and that 3-T1AM is produced extrathyroidally. Many researchers have reasoned that the aromatic L-amino acid decarboxylase (AADC) mediates TAM synthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC with several TH. In all tested conditions, AADC failed to catalyze the decarboxylation of TH. These in vitro observations are supported by the finding that 3-T1AM is also present in plasma samples of patients with AADC deficiency. In summary, 3-T1AM detection in serum using LC-MS/MS encounters preanalytical problems. The first MAb-based 3-T1AM CLIA is presented, which reliably quantifies 3-T1AM in human serum. AADC is likely not involved in TAM biosynthesis.

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Alothaim, Ibrahim. "Applications of LC-MS metabolomic profiling in inflammatory bowel disease and the development of derivatisation methods to enhance selective detection of certain compound classes". Thesis, University of Strathclyde, 2018. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=29531.

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Inflammatory bowel disease (IBD) is a chronic inflammation of all parts of the gastrointestinal tract and is represented by two major variations, ulcerative colitis (UC) and Crohn's disease (CD). It is diagnosed based on the pattern of inflammation. In order to understand the pathology of the disease better urine and saliva samples were collected from patients with IBD, patients in remission and a control group. The metabolomes of the urine and saliva samples were profiled by carrying out chromatography on a ZICpHILIC column in combination with high resolution mass spectrometry. It was possible to separate the different classes of urine samples on the basis of their metabolomic profiles by modelling with data using orthogonal partial least squares analysis (OPLSDA). A number of metabolites were found to vary between the three groups although the models for separation were weak. The OPLSDA models of the saliva data were much stronger and saliva analysis looks promising for diagnostic and prognostic purposes. Nine variables were able to discriminate the control and affected samples and these included four sphingosine bases. The analysis of short chain fatty acids (SCFAs) by LC-MS is a problem since they are too volatile to give good responses. SCFAs are potentially important makers for IBD. A quantitative LC-MS method for acetic, propionic, butyric and lactic acids was developed by carrying out derivatisation of the acids using a carbodimide to activate the acids and then reacting with dimethylaminophenylamine and separating the derivatives using HILIC chromatography. Quantification was carried out by using stable isotope dilution. The method was very sensitive but detection limits were set by the background contamination by the SCFAs rather than by absolute sensitivity. The method was applied to the urine samples and differences in acetate and butyrate were found between the affected and control samples. The microbiome plays a role in IBD and one marker for bacterial activity it the breakdown of dietary fibre in sugar monomers. Thus urine and saliva samples from controls and IBD samples were profiled using a reductive amination method previously developed. It was found that there were significant differences in the pattern of hexoses, pentoses and deoxy hexoses in the urine and saliva samples.

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Zamani, Leila. "Methods for structural studies of an antibody, screening metabolites in rat urine and analysis of spent cell cultivation media using LC/ESI-MS and chemometrics". Doctoral thesis, Stockholms universitet, Institutionen för analytisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-28921.

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This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs. Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control. In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.

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37

Goodpaster, Aaron M. "Statistical Analysis Methods Development for Nuclear Magnetic Resonance and Liquid Chromatography/Mass Spectroscopy Based Metabonomics Research". Miami University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=miami1312317652.

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38

Kim, Dong Hyun. "Investigation of HIV anti-viral drug effect on HPV16 E6 expressing cervical carcinoma cells using advanced metabolomics methods". Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/investigation-of-hiv-antiviral-drug-effect-on-hpv16-e6-expressing-cervical-carcinoma-cells-using-advanced-metabolomics-methods(d52b3b66-2a7b-4577-a334-b74bc12b27cc).html.

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Metabolomics approaches have recently been used to understand the complex molecular interactions of biological systems. One popular area in which these methods are being developed is to understand the biochemical changes during abiotic and biotic stresses; for example, how a cell may respond to a drug. Since metabolites are the end products of gene expression, these can be used to indicate the result of the activities and interaction of the cell or organism with its environment. The investigation of the level and compositional changes of metabolites against metabolic stresses such as chemotherapeutic treatment (drug exposure) are required to understand more fully abiotic perturbation to biological systems. The aim of this project was to understand the metabolic effect that the anti-viral drugs indinavir and lopinavir (currently used by HIV patients) have on HPV-related cervical cancer cell lines by measuring changes in metabolism using a wide range of analytical techniques; including Fourier transform infrared (FT-IR) and Raman spectroscopies, and gas and liquid chromatography-mass spectrometry (GC and LC-MS). The analyses and interpretation of the large volumes of complex multidimensional data generated by metabolomics approaches were performed with a combination of multivariate data analysis techniques such as principal components analysis (PCA) and canonical variates analysis (CVA), as well as univariate approaches such as N-Way analysis of variance (ANOVA). By combining biochemical imaging, metabolite fingerprinting and footprinting, and metabolite profiling, with multi- and uni-variate analyses, the actions and effects of the anti-viral drugs were investigated. FT-IR spectroscopy was initially used to generate global biochemical finger- and foot-prints, and Raman spectroscopy was employed to investigate intracellular distribution of metabolites, and other cellular species, as well as the localisation of drug molecules within cells. FT-IR spectroscopy ascertained that the intra- and extra-cellular metabolomes were being directly influenced in a fashion that correlated with increasing anti-viral dosing; these effects were phenotypic rather than measurements of the drug level. Raman imaging spectroscopy indicated that the indinavir but not lopinavir was being compartmentalised within the cell nucleus, but only in HPV early protein 6 (E6) expressing cells. This observation was further confirmed by fractionation of cell samples into nuclear and cytoplasmic fractions and assessing the indinavir concentrations via LC-MS. Finally, LC-MS and GC-MS metabolite profiling were employed to investigate changes in the intracellular metabolome in response to the anti-viral compounds across a range of physiologically relevant concentrations and in the presence and absence of the E6 oncoprotein. General effects of both anti-viral compounds included the regulation of metabolites such as glutathione, octenedionoic and octadecenoic acids, which may be involved in stress related responses, reduced levels of sugars and sugar-phosphates indicating a potential arrest of glycolysis, and reduced levels of malic acid indicating potential decreased flux into the TCA cycle; all indicating that central metabolism was being reduced. Finally, LC-MS based quantification indicated that in the presence of E6, lopinavir was actively removed from the cell, whereas the indinavir intracellular concentration increased concomitantly with the level of dosing. These investigations have revealed that metabolomics approaches are an apt tool for the study of anti-viral effects within cell cultures, but improvements need to be made with respect to the major limitation of metabolite identification.

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39

Ullah, Shahid. "Improved analytical methods for perfluoroalkyl acids (PFAAs) and their precursors – a focus on human dietary exposure". Doctoral thesis, Stockholms universitet, Institutionen för tillämpad miljövetenskap (ITM), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-93719.

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Per- and polyfluoroalkyl substances are a large group of global environmental contaminants. They can be divided into two sub-groups, 1) perfluoroalkyl acids (PFAAs) and 2) so called precursors, i.e. compounds that can potentially be transformed to form PFAAs. PFAAs are today ubiquitous in wildlife and humans. Food and drinking water are assumed to be the dominant human exposure pathways for PFAAs. The main aim of this doctoral thesis was to develop highly sensitive and fully validated analytical methods for the determination of a range of PFAAs and selected precursors in dietary samples. The methods were based on liquid chromatography coupled to mass spectrometry. Samples were extracted by solvent extraction followed by a cleanup step employing solid phase extraction. The cleanup step could at the same time be used as a fractionation of ionic PFAAs and neutral precursors. Paper I and II describe the development of methods for simultaneous analysis of three groups of PFAAs including perfluoroalkyl phosphonic acids (PFPAs) in drinking water and food. Methyl piperidine was used as ion pairing agent, leading to highly sensitive analysis of PFPAs. A first screening of tap water samples and different food items revealed that human dietary exposure to PFPAs in Europe is currently not of concern. A novel method for simultaneous analysis of perfluoroalkyl carboxylic acids (PFCAs) and polyfluoroalkyl phosphate esters (PAPs) in food and packaging materials is described in paper III. Targeted food samples and their packaging were analyzed. The results showed that PAPs may contribute to human exposure to PFCAs. In paper IV temporal trends (1991-2011) of perfluorooctane sulfonic acid (PFOS) and its precursors in herring were investigated. Rapidly decreasing trends were found for precursors, whereas PFOS did not show a significant change over time. Precursors in fish may have played an important role for human exposure to PFOS in the 1990s but are probably negligible today.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

PERFOOD project (KBBE-227525)

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40

Tollbäck, Johanna. "New methods for determination of airborne pollutants : Focus on tetrabromobisphenol A, organophosphate triesters and polycyclic aromatic hydrocarbons". Doctoral thesis, Stockholms universitet, Institutionen för analytisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-26700.

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The work presented in this thesis concerns the development and evaluation of new methods of sampling and analysis of organic pollutants in the indoor and outdoor environment. In Paper I, the development of a new method was reported for the determination of the brominated flame retardant tetrabromobisphenol A (TBBPA) in air using sampling with glass fiber filter and polyurethane foam (PUF), ultrasonic solvent extraction and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC-ESI/MS). The MS fragmentation mechanism of TBBPA was thoroughly investigated and different acquisition modes were evaluated to achieve the most sensitive and selective detection. In Papers II and III, the potential use of Empore SPE membranes was evaluated for air sampling of volatile, semi-volatile and particle-associated organic compounds. Breakthrough studies conducted for 24h at air flows of 10- 20 L/min showed that the SPE membranes efficiently retains volatile and semi-volatile organophosphate esters and particles >10 nm. Effort was invested in the development of fast and environmental friendly methods, with low cost, for sample clean up and analysis. In Paper II, the sample preparation technique was dynamic solvent extraction with methanol coupled to LC-ESI/MS. The total run time per sample, including both extraction and separation, was less than 34 min, consuming only 1.6mL methanol. In Paper III, efficiency of selective extraction of polycyclic aromatic hydrocarbons from particulate matter sampled with Empore SPE membranes, using dynamic subcritical water extraction (DSWE) was investigated. Acceptable recoveries of the investigated compounds from reference material (SRM 1649a) were achieved. In Paper IV, the application of dynamic solid phase micro-extraction (SPME) air sampling was evaluated using, gas chromatography/positive ion chemical ionisation (GC/PICI) and tandem-MS detection for the determination of organophosphate esters in work environment.

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41

Spáčil, Zdeněk. "Mass Spectrometry of Biologically Active Small Molecules : Focusing on polyphenols, alkaloids and amino acids". Doctoral thesis, Stockholms universitet, Institutionen för analytisk kemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-33233.

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The foci of this dissertation are on advanced liquid chromatography (LC) separation and mass spectrometry (MS) techniques for the analysis of small bioactive molecules. In addition to discussing general aspects of such techniques the results from analyses of polyphenols (PPs), alkaloids and amino acids published in five appended studies are presented and discussed. High efficiency and well understood principles make LC the method of choice for separating analytes in many kinds of scientific investigations. Moreover, when LC is coupled to an MS instrument, analytes are separated in two stages: firstly they are separated and pre-concentrated in narrow bands using LC and then separated according to their mass-to-charge (m/z) ratios in the MS instrument. Some MS instruments can provide highly accurate molecular weight measurements and mass resolution allowing identification of unknown compounds based purely on MS data, thus making prior separation unnecessary. However, prior separation is essential for analyzing substances in most complex matrices – especially useful is the ultra-high performance LC (UHPLC). The advantages of using UHPLC rather than HPLC for the analysis of PPs in tea and wine were evaluated in one of the studies this thesis is based upon. The phenolic composition of red wine was also examined, using a novel LDI technique, following solid phase extraction (SPE). A class of small aromatic molecules (medicinally important alkaloids) also proved to be amenable to straightforward analysis, by thin layer chromatography (TLC) work-up followed by LDI-MS. Finally, a LC-MS method for monitoring neurotoxins (β-N-methyl-amino-L-alanine and 2,3-diaminobutyric acid) in complex biological matrices was developed and applied. Overall, the studies show that careful attention to the physicochemical properties of analytes can provide insights that can greatly facilitate the development of alternative methods to analyze them, e.g. by LDI.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript.

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42

Höfig, Carolin [Verfasser], Werner [Akademischer Betreuer] Kloas, Josef [Akademischer Betreuer] Köhrle y Dagmar [Akademischer Betreuer] Führer-Sakel. "Establishment, validation and application of immunological and LC-MS/MS-based detection methods to study the role of human aromatic L-amino acid decarboxylase as an enzyme potentially involved in thyronamine biosynthesis / Carolin Höfig. Gutachter: Werner Kloas ; Josef Köhrle ; Dagmar Führer-Sakel". Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://d-nb.info/1029763844/34.

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43

Carvalho, Jose Joao. "Immunochemical and chromatographic methods for two anthropogenic markers of contamination in surface waters". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16420.

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Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt.
Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.

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Utami, Wahyu. "Ion pairing LC-MS/MS method for analysis of intracellular phosphorylated metabolites". Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/30566/.

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Nucleoside analogues have been extensively used in medication. The nucleoside analogue cordycepin is the principal bioactive compound found in the caterpillar fungi (Cordyceps and Ophiocordyceps). It has been shown to have biological activity, including anti-inflammatory, immunomodulatory and anti-proliferative activity in many kinds of malignant cells. Intracellular drug interactions at the nucleotide level can be explained by understanding the intracellular metabolism of nucleoside analogues as well as their plasma metabolism since their efficacy of therapy or toxicity does not associate with the plasma level of nucleoside. Therefore, investigation of the metabolism of nucleoside analogues is required for a full understanding of their pharmacological activity and toxicity. For that reason, here an ion pairing LC-MS/MS method has been developed for quantitative analysis of the nucleoside analogue cordycepin and the metabolites and its application to cell culture, using in vitro and in vivo studies. Several HPLC parameters and extraction techniques have been optimised, followed by optimisation of the mass spectrometry method by examining the fragmentation of nucleotides. The method was then validated and applied to study the metabolism of cordycepin in vitro and in vivo, to investigate the effect of the cordycepin treatment with or without pentostatin on the intracellular level of endogenous nucleotides, and to examine the intracellular metabolism of nucleoside analogue 4-thiouridine and the effect of its metabolite on the metabolic balance of adenine and uridine nucleotides. The study on the intracellular metabolism of cordycepin in MCF7 and HeLa cells shows that cordycepin was rapidly metabolized into the deaminated form by adenosine deaminase (ADA) in culture medium as well as in cancer cells; therefore combination with pentostatin, an ADA inhibitor, resulting in the highly accumulated phosphorylated metabolite intracellularly. In contrast, cordycepin in C. militaris extracts showed much lower degradation in non-heat-treated serum compared with pure cordycepin that indicates a strong evidence of the presence of a deaminase inhibitor in the extract of Cordyceps. Moreover, the determination of concentrations of cordycepin and the metabolites in the plasma and liver of rats dosed with cordycepin proves that the half-life of cordycepin and its metabolites are very short in the plasma; nevertheless they are accumulated in the liver with repeated administration. Treatment using cordycepin initially caused an increase in the intracellular concentrations of nucleoside triphosphate, but in the long term, the active metabolite of cordycepin likely induced a long term change in the cell resulting in a drop in nucleotide levels. Pentostatin on its own reduced nucleoside triphosphates levels in the long term and combination with cordycepin increased the effect of cordycepin on nucleotide concentrations. High levels of the accumulated cordycepin triphosphate led to a massive decline in nucleotide levels. A study on the intracellular metabolism of nucleoside analogue 4-thiouridine has shown that generally the uptake of 4-thiouridine into NIH 3T3 cells was fast and the phosphorylated metabolite rapidly was developed only after two min labelling. However, it was also shown that its phosphorylation was not very efficient, but the level of the phosphorylated metabolite increased in serum-stimulated cells likely because the enzyme was upregulated in the presence of growth factor. Moreover, the present study provides additional evidence that 4-thiouridine and its metabolite have no adverse effect on the metabolic balance of adenine and uridine nucleotides. This study confirms that pharmacological activity of nucleosides analogues and their cytotoxicity highly rely on the accumulation of their phosphorylated metabolites. Consequently, the activity and the level of the enzymes involved in their metabolism are highly influential on their pharmacological action as well as their toxicity.

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Yacoub, Kimberly. "Development of ESI-LC-MS Method for Drug Analysis". Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1524040258129489.

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Becker, Matthias. "LC-MS/MS-Methoden zur Rückstandsanalyse von Penicillinen, Cephalosporinen und Aminoglycosid-Antibiotika". [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974085324.

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47

Persson, Josefin. "Development and evaluation of methods for analysis of TBECH and HBCD using HRGC/HRMS and HPLC/MS/MS". Thesis, Örebro University, School of Science and Technology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-7695.

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The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are simply mixed with material and are most likely to leach out in the environment, because of non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-, γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γ-HBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to and bioaccumulate in water-living animals.

In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How much of the TBECH isomers that were recovered after applying existing extraction and clean-up procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts to three different whale samples indicate severe evaporation losses and possibly photolytic degradation. None of the four enantiomers were detected in the three whale samples. For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δ- and ε- HBCD yielding good chromatography and sensitivity. However, due to technical difficulties during the time-period of this project, no whale samples could be analysed for HBCD on UPLC/MS/MS.

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Tang, Jennifer Huiqin. "DEVELOPMENT OF A LC/MS/MS ENZYME METHOD FOR N8 - ACETYLSPERMIDINE MEASUREMENTS IN ENZYME ASSAYS". Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/607.

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This thesis describes the development of a way to study the N8 - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety. In this study, HeLa cells were cultured and extracted to yield a crude N8 - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N8 - acetylspermidine and the increase of spetmidine, N8 -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N8 -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity. The quantitation of N8 -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d6- and dg-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N8 - acetylspermidine deacetylase enzyme activity.

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GIACCONE, VITA. "Identificazione e quantificazione delle Micotossine prodotte da Alternaria in alimenti e mangimi, mediante LC-MS/MS e LC/HRMS". Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2022. http://hdl.handle.net/11380/1276559.

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Le micotossine sono contaminanti significativi negli alimenti e nei mangimi e sono causa di gravi effetti tossici sulla salute umana e animale. Diversi studi hanno dimostrato l’esistenza di una moltitudine di metaboliti fungini che contaminano alimenti e mangimi. Recentemente, le autorità sanitarie internazionali hanno espresso preoccupazione per la presenza di micotossine "emergenti" negli alimenti. Tra le specie fungine responsabili della produzione di tali metaboliti, la specie Alternaria ha la capacità di produrre più di 70 tossine, come Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), presenti nei cereali, in prodotti a base di pomodoro, nell’olio d'oliva, nella frutta e nella verdura fresca. Queste micotossine sono state recentemente oggetto di pareri scientifici dell'Autorità Europea per la Sicurezza Alimentare (EFSA), che ha espresso preoccupazione per la scarsa disponibilità di dati sull'incidenza negli alimenti destinati al consumo umano e animale. Esiste, infatti, un alto grado di incertezza legato alla rappresentatività degli alimenti attualmente testati. Inoltre, la mancanza di informazioni sul metodo analitico utilizzato contribuisce ulteriormente a creare incertezza sui livelli di tossine da Alternaria. Sono necessari ulteriori studi per aggiornare la valutazione del rischio da parte dell'EFSA e, pertanto, l'obiettivo specifico di questo progetto era incentrato sullo sviluppo di metodi LC-MS/MS e LC/HRMS accurati per l'analisi delle tossine da Alternaria. Il progetto è stato sviluppato in collaborazione con l'Istituto Zooprofilattico Sperimentale della Sicilia. Il metodo LC-MS/MS è stato utilizzato per l'analisi target delle micotossine emergenti nelle matrici alimentari. In particolare sono state svolte le seguenti attività: • ottimizzazione dei parametri del metodo per lo screening simultaneo di Alternariol (AOH), Alternariol monometiletere (AME), Acido tenuazonico (TeA), Ocratossine e Zearalenone (ZEA), Fumonisina B1, B2, B3, tossina T-2, HT-2 tossina e deossinivalenolo (DON) (vomitossina); • sviluppo di un protocollo di preparazione del campione, da applicare a cereali e frutta secca; il protocollo di preparazione del campione si è basato su una doppia estrazione (estrazione liquida) e purificazione mediante estrazione in fase solida (SPE). • validazione del metodo secondo le linee guida comunitarie (Decisione della Commissione 2002/657/CE); il metodo è stato ottimizzatoo valutando i seguenti parametri: specificità, precisione, linearità strumentale, recupero, limite di decisione (CCα), capacità di rilevamento (CCβ), effetto matrice e robustezza. La cromatografia accoppiata alla spettrometria di massa ad alta risoluzione (UHPLC-Q-HRMS) è stata utilizzata per la determinazione, sensibile e specifica, delle tossine da Alternaria. Le prestazioni ottenute sono state confrontate con quelle ottenute mediante cromatografia liquida ad alte prestazioni. Tutti e due i metodi hanno mostrato una buona linearità e ripetibilità. Si dice che lo spettrometro di massa Q-Exactive sia più adatto per il rilevamento in tracce rispetto ai metodi MS/MS basati sul triplo quadrupolo, perché, nonostante abbia prestazioni comparabili, ha una migliore selettività. Il progetto di ricerca avrebbe dovuto fornire un'ampia indagine sul contenuto emergente di micotossine in vari prodotti alimentari, contribuendo a una stima dell'esposizione al rischio di micotossine. Le difficoltà legate alla pandemia in corso hanno reso difficile la ricerca, inizialmente prevista in questo progetto. Nonostante tutto, lo scopo di questo lavoro è stato colmare il vuoto di dati e fornire informazioni rilevanti per migliorare la sicurezza dei prodotti locali.
Mycotoxins are significant contaminants in food and feeds. These molecules have demonstrated serious effects in both human and animal health. Different studies showed the presence of a multitude of fungal metabolites that contaminate food and feed. Recently, the international health authorities have expressed concerns about the presence of "emerging" mycotoxins in foodstuffs. The term “emerging mycotoxins” is commonly referred to those compounds that are currently under the spotlight of the scientific community and the policy-makers, due to their toxicological profile. Among the fungal species responsible for the metabolite production, Alternaria species have the ability to produce more than 70 toxins, such as Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), found in cereals, tomato products, olive oil, fresh fruits and vegetables. These mycotoxins have been recently covered by scientific opinions from the European Food Safety Authority (EFSA), who has expressed concern about the low availability of incidence data in food for human and animal consumption. There is, indeed, a high degree of uncertainty related to the representativeness of the food currently tested because it contains too many inaccuracies. Furthermore, the lack of information on the analytical method used further contributes to the uncertainty of the reported Alternaria toxin levels. More comprehensive studies are required to update the risk evaluation by the EFSA and, therefore, the specific aim of this project was focussed on the development of accurate LC-MS/MS and LC/HRMS methods for the analysis of emerging Alternaria toxins. The protocol was employed in collaboration with the Istituto Zooprofilattico Sperimentale della Sicilia. LC-MS/MS method was used for the target analysis of emerging mycotoxins in food matrices. In particular, the following activities were carried out: • optimisation of the method parameters for simultaneous screening of Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA), Ochratoxins and Zearalenone (ZEA), Fumonisin B1, B2, B3, T-2 toxin, HT-2 toxin and deoxynivalenol (DON) (vomitoxin); • development of a sample preparation protocol, apply to cereals and dry fruits; the sample preparation protocol was based on a double extraction (Liquid extraction) and purification through solid-phase extraction (SPE). • validation of the method according to EU guidelines (Commission Decision 2002/657/EC); the method was performed by evaluating the following parameters: specificity, precision, instrumental linearity, recovery, decision limit (CCα), detection capability (CCβ), matrix effect and ruggedness. The ultrahigh-performance chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-HRMS) was applied to the sensitive and specific determination of the emerging Alternaria toxins. Performances were compared to those obtained by high-performance liquid chromatography detection. All two methods showed good linearity and repeatability. The Q-Exactive mass spectrometer is said to be better suitable for trace detection than state-of-the-art MS/MS methods based on the triple quadrupole, because, despite having performance comparable, it has better selectivity. The research project should have provided a broad investigation of the emerging mycotoxin content in various food products, contributing to an estimate of mycotoxin risk exposure. The difficulties linked to the pandemic in progress made difficult research, initially planned within this project. Despite everything, the purpose of this work was to fill the data gap and provide relevant information to improve the safety of local products.

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Pantiru, Monica Elena. "Entwicklung einer LC-MS-MS-Methode zur Analytik polarer Pflanzenschutzmittel-Wirkstoffe und ihrer Metabolite in Erntegütern". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972779329.

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