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Anticarcinogenic compounds in watercressRose, Peter Colin January 2001 (has links)
No description available.
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Erhebung pharmakokinetischer Daten von Cisaprid, SC-72393, Haloperidol, Linezolid, Methotrexat und Ketoprofen nach Methodenentwicklung und Validierung durch LC-MS/MS-Detektion / Enquiry of pharmacokinetic parameters of Cisapride, SC-72393, Haloperidol, Linezolid, Methotrexat and Ketoprofen after method development and validation by LC-MS/MSSteinhauer, Sven January 2002 (has links) (PDF)
Zur Bestimmung von geringen Wirkstoffkonzentrationen in biologischen, speziell human-biologischen Matrizes wie Blut, Urin oder Mikrodialysat bedarf es einer Analysentechnik, die den Wirkstoff mit einem Höchstmaß an Selektivität, Spezifität und Präzision bestimmen kann. Daneben muß die verwendetete Methode eine hohe Geschwindigkeit aufweisen und sehr robust sein, da bei der heutigen marktwirtschaftlichen Lage Analysensysteme eine optimale Auslastung erfahren müssen. Aus diesem Grund ist der Umbau oder die Umstellung der Methode von einem zum anderen Wirkstoff ohne nennenswerten Zeitverlust ein maßgeblicher Faktor. Als Technik, die diese Anforderungen optimal erfüllt, hat sich in den letzten Jahren die LC-MS/MS-Technik etabliert. Sie ist den bislang überwiegenden Methoden, wie GC-MS-Techniken oder HPLC-UV-Detektion bzw. Fluoreszenztechniken in Bezug auf die oben genannten Parameter deutlich überlegen. In der vorliegenden Arbeit wurden für die Wirkstoffe Cisaprid, SC-72393, Haloperidol, Linezolid, Methotrexat und Ketoprofen LC-MS/MS-Methoden entwickelt oder via unabhängiger Laborvalidierung auf die lokalen Gegebenheiten transferiert und zur Bestimmung pharmakokinetischer Parameter zur Anwendung gebracht. Ziel der Methodenentwicklung war es die hohe Selektivität und Empfindlichkeit des Detektors zu nutzten, um bei geringen Probenvolumina eine Bestimmungsgrenze zu erreichen, die es ermöglichte ausreichend viele Meßwerte zu bestimmen, um die pharmakokinetischen Parameter der Wirkstoffe zu berechnen. Zusätzlich wurde eine Maximierung des Probendurchsatzes und eine Minimierung des personellen und materiellen Aufwandes angestrebt ohne dabei einen Qualitätsverlust der Methode zu erleiden. Eine gelungene Methoden-entwicklung bedurfte daher der Optimierung der Probenaufarbeitung, die sich neben den chemisch-physikalischen Eigenschaften des Wirkstoffes hauptsächlich an der Menge der zur Verfügung stehenden Probe orientierte. Das chromato-graphische System hingegen hing weitestgehend von den chemischen Eigenschaften des Analyten und von den massenspektroskopischen Bedingungen ab, die verdampfbare Puffer im Fließmittel erforderten. Diese drei zu optimierenden Teilbereiche, die miteinander interagieren, wurden jeweils sorgsam aufeinander abgestimmt, um eine Methode zu entwickeln, die die zu erwartenden Wirkstoffkonzentrationen in der jeweiligen Matrix sicher und robust bis hin zum Quantifizierungslimit bestimmen konnte. / In order to quantify low drug concentrations in biological, specifically human matrices as blood, urine or microdialysate it is indispensable to use a technique, that is able to evaluate the drug with a maximum of selectivity, specifity and precision. In addition the used method needs to be fast and robust to meet todays economical requirements. For this reason the used equipment has to be exchangeable within the used methods without any time interuptions worth mentioning. The LC-MS/MS-technique, which has been established as routine technic over the last decade fulfils these requirements. It is superior with regards to selectivity, specifity and precision over methods such as GC-MS or HPLC with UV- or fluorescense-detection. In this thesis method development, validation or independent laboratory validation was done with LC-MS/MS on Cisapride, SC-72393, Haloperidol, Linezolid, Methotrexat and Ketoprofen to determine their concentrations and to calculate pharmakokinetic parameters. The objective of the method development was to use the high selectivity and sensetivity of the detector to reach a low quantification limit with the limited specimen volumes given. Besides that a maximum throughput of specimens with a minimum of personal expense was aspired without decreasing the quality of the produced data by taking into account the phamakokinetic parameters to be determined. Therefore an optimum of the work-up procedure as well as the chromatographic and the mass spectrometric conditions has to be developed for the individual drugs with their physicochemical properties. Limitations were frequently given by the availability of the specimen volume. Those three parts, which interact with each other were carefully coordinated to develop a method that quantifies the specific drug concentrations in the individual matrix with good sensitivity and robustness down to the limit of quantification.
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Development of practice-oriented LC-MS/MS methods for the determination of important drugs and their application for building PK/PD concepts / Die Verwendung von praxisorientierten LC-MS/MS Methoden für die Bestimmung von wichtigen Arzneistoffen und ihre Verwendung zur Erstellung von PK/PD KonzeptenRodamer, Michael January 2011 (has links) (PDF)
In this thesis eight robust and reliable LC-MS/MS methods were developed and validated to analyze atorvastatin, clopidogrel, furosemide, itraconazole, loratadine, naproxen, nisoldipine and sunitinib in human plasma. The active metabolites 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, hydroxyitraconazole, descarboethoxy-loratadine, 4-hydroxynisoldipine and N-desethylsunitinib were also included in the corresponding methods. Due to the different physical, chemical and pharmacokinetic properties of the analytes a wide spectrum regarding sample preparation techniques, chromatography and mass spectrometric detection was covered. Protein precipitation methods were developed for furosemide, itraconazole, naproxen, nisoldipine and sunitinib. Liquid-liquid extraction methods were developed for atorvastatin, clopidogrel and loratadine. Criteria to choose protein precipitation or liquid-liquid extraction were the final plasma concentrations of the drugs, which are mainly dependant on the dose, bioavailability and t1/2 and of course cost-effectiveness. Altogether, the methods have a concentration range from 0.001 ng/mL (LLOQ of clopidogrel) to 50000 ng/mL (highest calibration point for naproxen), covering 5 x 107 orders of magnitude. The runtime of the methods ranged from 2 to 4 minutes, facilitating a high sample throughput. All developed methods were validated according to recent guidelines as they were used to analyze sampes from clinical trials. Excellent linearity, intra-day and inter-day precision and accuracy were observed in the validated calibration ranges. Hemolyzed, lipemic and different batches of human plasma as well as sample dilution did not affect the determiantion of the analytes. Clopidogrel, loratadine, nisoldipine and sunitinib and if available their metabolites were subjected to a matrix effect test, resulting in no influence of different batches of human plasma on the analytical methods. Noteworthy is clopidogrel that shows a slight effect on one of the two used mass spectrometers. However, that effect was reproducible and did therefore not affect clopidogrel determination. No evidence of instability during chromatography, extraction and sample storage processes for all analytes except 4-hydroxyatorvastatin was found, for which a significant decrease was observed after three months. During incurred sample reanalysis of study samples 95 % of the samples were within ±15 % with respect to the first analysis. Moreover, the atorvastatin, loratadine and clopidogrel method were compared on two generations of triple quadrupole mass spectrometers, the API 3000™ and the API 5000™. The new ion source and the changes in the ion path of the API 5000™ provided higher sensitivity, the extend depending on the substance. However, the API 3000™ had very good precision in the performed system comparison. The validated methods showed excellent performance and quality data during routine sample analysis of eight clinical trials. Moreover, they are suitable for high sample throughput due to their short run times. / In dieser Dissertation wurden acht robuste und verlässliche LC-MS/MS-Methoden zur Analyse von Atorvastatin, Clopidogrel, Furosemid, Itraconazol, Loratadin, Naproxen Nisoldipin und Sunitinib in Humanplasma entwickelt und validiert. Außerdem enthalten die Methoden die aktiven Metaboliten 2-Hydroxyatorvastatin, 4-Hydroxyatorvastatin, Hydroxyitraconazol, Descarboethoxyloratadin, 4-Hydroxynisoldipin und N-Desethylsunitinib. Wegen der unterschiedlichen physikalischen, chemischen und pharmakokinetischen Eigenschaften der Analyten, deckt diese Arbeit ein weites Spektrum bezüglich Probenaufarbeitung, Chromatographie und Massenspektrometrie ab. Präzipitationsmethoden wurden für Furosemid, Itraconazol, Naproxen, Nisoldipin und Sunitinib entwickelt. Flüssig-flüssig-Extraktionen wurden für Atorvastatin, Clopidogrel und Loratadin entwickelt. Kriterien für die Auswahl von Präzipitation oder Extraktion waren die erwartete Plasmakonzentration, die im Wesentlichen von der Dosis, Bioverfügbarkeit und Halbwertszeit abhängig ist, und natürlich Kosteneffektivität. Insgesamt erstrecken sich die Methoden über einen Kalibrierbereich von 0.001 ng/mL (LLOQ von Clopidogrel) bis zu 50000 ng/mL (HLOQ von Naproxen), das entspricht 5x107 Größenordnungen. Die Laufzeiten pro Probe liegen im Bereich von zwei bis vier Minuten, was einen sehr hohen Probendurchsatz ermöglicht. Alle in dieser Arbeit entwickelten Methoden wurden gemäß aktueller Richtlinien (FDA, GLP) validiert und verwendet um Proben aus Pharmakokinetikstudien zu analysieren. Ausgezeichnete Linearität, Präzision und Genauigkeit zeichnen diese Methoden aus. Hämolysiertes, lipämisches und verschiedene Batches von Humanplasma, sowie Vorverdünnung hatten bei keiner Methode Einfluss auf die Bestimmung der Analyten. Clopidogrel, Loratadin, Nisoldipin und Sunitinib und gegebenenfalls deren Metabolite wurden einem Matrix-Effekt-Test unterzogen. Dabei wurde festgestellt, dass keine der Methoden durch die Probenmatrix beeinflusst wurde. Erwähnenswert ist Clopidogrel, da an einem der Massenspektrometer ein leichter Effekt beobachtet werden konnte, der sich auf alle untersuchten Matrices gleich auswirkte und somit keinen Einfluss auf die gesamte Methode hatte. Weiterhin fand sich bei keiner der untersuchten Substanzen ein Hinweis auf Instabilität während der Probenlagerung, -aufarbeitung und -messung, außer bei 4-Hydroxyatorvastatin, dessen Konzentration nach drei Monaten signifikant abnahm. Während der Reanalyse von Studienproben (incurred samples) lagen über 95 % der Proben innerhalb von ±15 % im Vergleich zur ersten Messung. Außerdem wurden die Methoden zur Bestimmung von Atorvastatin, Loratadin und Clopidogrel an zwei Generationen von Massenspektrometern verglichen, nämlich dem API 3000™ und dem API 5000™. Die neue Ionenquelle und die Verbesserungen im Ionenpfad beim API 5000™ ermöglichten - abhängig von der analysierten Substanz - höhere Sensitivität. Allerdings konnte das API 3000™ bei den durchgeführten Experimenten mit einer hohen Präzision aufwarten. Die validierten Methoden zeigten im Alltagbetrieb bei der Messung von acht klinischen Studien hervorragende Performance und Qualitätsdaten. Darüber hinaus sind die Methoden aufgrund ihrer kurzen Laufzeiten ideal für Messungen die einen hohen Probendurchsatz erfordern.
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Utveckling och validering av en LC-MS/MS metod för kvantifiering av clopidogrel och dess metabolit i plasmaShamon, Doreen-Marie January 2010 (has links)
<p>Clopidogrel is an antiplatelet substance that prevents blood coagulation in the arteries. It is an inactive pro drug that becomes activated after first-pass metabolism by the liver. The active metabolite of clopidogrel is 2-oxoclopidogrel, which is unstable therefore pharmacokinetic data is obtained by measuring the inactive metabolite clopidogrel acid in plasma. Clopidogrel is taken orally in tablet form. The aim of this project was to develop a LC-MS/MS method for quantification of clopidogrel and its metabolite in plasma.</p><p> </p><p>The method has been developed by optimizing the sample preparation. Different extraction procedures and extraction columns were tested, for example, by changing the extraction column from a C8 silica sorbent to Oasis HLB (a polymer sorbent). Different internal standards were evaluated as a result of discovering the signal suppression of the previous internal standard clopidogrel acid. Flupentixol was found to be the best candidate.</p>
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Dosage de biomarqueurs dans les fluides biologiques par chromatographie liquide ou électrophorèse capillaire couplée à la spectrométrie de masseMartin, Gaëlle 16 December 2009 (has links)
La découverte, la caractérisation et le dosage de biomarqueurs ont permis douvrir de
nouveaux horizons tant en thérapeutique que dans létablissement dune médecine
personnalisée. Dans un contexte danalyse en milieu complexe, lapplication de
techniques fiables, sensibles, exactes et de haute résolution est de rigueur. Dans ce but,
le couplage de la spectrométrie de masse à une technique séparative telle que la
chromatographie liquide (LC-MS) ou lélectrophorèse capillaire (CE-MS) offre des
potentialités intéressantes.
Une méthode de dosage de la cysdopa, un marqueur du mélanome, dans le
plasma par LC-MS a été mise au point. Une étape préliminaire dextraction en phase
solide sur un support hybride alliant des interactions de type hydrophobe, hydrophile et
échangeurs de cations a conduit à lobtention dexcellents taux de récupération. Les
conditions chromatographiques assurant la séparation de la cysdopa des composés
endogènes susceptibles dinterférer et les paramètres de la détection par spectrométrie
de masse en tandem ont été optimisés. La présence déventuels effets de la matrice a
été investiguée en détails. Une étude de la stabilité de la cysdopa dans les différentes
conditions opératoires a été réalisée répondant ainsi aux normes préscrites par la FDA.
Ensuite, une validation complète de la méthode en milieu plasmatique selon lapproche
faisant appel aux profils dexactitude sur une gamme de concentration allant de 1,6 à
200 ng/ml a été menée avec succès démontrant ladéquation de la méthode. Enfin, une
étude pilote ainsi quune validation en cours détude ont permis de tester cette méthode
de dosage.
La seconde technique mise en oeuvre consiste en lapplication du couplage CE-MS
à lanalyse de lhepcidine, un petit peptide régulant le métabolisme du fer. Les conditions
électrophorétiques assurant la séparation de ce peptide des constituants dun mélange
type ont été déterminées. Le traitement de la paroi interne du capillaire et laddition
dune cyclodextrine nont pas permis dans la présente application daméliorer la
sélectivité et lefficacité des pics. Lutilisation dun électrolyte de haute force ionique
associé à laddition dun modificateur organique a par contre offert les meilleures
performances. Une approche multivariée en deux temps (criblage puis modélisation) a
été appliquée pour loptimisation des paramètres CE-MS. Les effets principaux du voltage
du capillaire et de la pression du gaz nébulisant se sont révélés être significatifs tout
comme leffet quadratique de ce dernier ainsi que les interactions entre la concentration
en électrolyte et le voltage du capillaire dune part et la pression du gaz nébulisant
dautre part. Finalement, la sensibilité en terme de rapport signal/bruit a été comparée
en CE-UV et CE-MS établissant les potentialités du couplage CE-MS pour lanalyse de
peptides.
Discovery, caracterization and determination of biomarkers provide new
perspectives in therapeutics and establishment of personnal medecine. Analysis in
complex matrix requires reliable, sensitive, accurate and high resolution techniques. To
achieve this, coupling of mass spectrometry with separative techniques such as liquid
chromatography (LC-MS) or capillary electrophoresis (CE-MS) offer interesting
potentialities.
A method for the determination of cysdopa, a melanoma biomarker, in human
plasma was developed using LC-MS. Solid phase extraction in mixed mode, combining
hydrophobic, hydrophilic and cation exchange interactions led to excellent recovery. The
chromatographic conditions ensuring separation of cysdopa from potential by interfering
endogenous compounds and tandem mass spectrometry detection parameters were
optimised. The presence of matrix effect was investigated in detail. A full stability study
was then performed according to FDA requirements. Finally, a complete validation of the
method in plasma following the approach of accuracy profiles over a concentration range
from 1,6 to 200 ng/ml was successfully performed, demonstrating adequacy of the
developed method. A pilot study and an in-study validation were finally perfomed to test
this assay method.
The second implemented technique consists in the application of CE-MS coupling
to hepcidin analysis, a small peptide regulating iron metabolism. Electrophoretic
conditions ensuring separation of hepcidin from model peptides have been determined.
Resolution and/or efficiency were not improved by using coated capillaries or by adding a
cyclodextrin. A high ionic strength electrolyte associated to the addition of an organic
modifier provided the best performance. A multivariate approach (screening and
modeling) was applied to the optimisation of CE-MS parameters. The principal effet of
capillary voltage and nebulizing gas pressure were significant as well as the quadratic
effet of the last parameter and the interactions of electrolyte concentration and, for one
part, capillary voltage and, for another part, nebulizing gas pressure. Finally, sensitivity
with respect to signal/noise ratio was compared between CE-UV and CE-MS, assessing
potentialities of CE-MS for peptide analysis.
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Determination of Macrolide and Lincoamide Antibiotic in Fish Muscle by High Performance Liquid Chromatography- Tandem Mass SpectrometryChen, Yu-chieh 27 August 2010 (has links)
The main research of this thesis includes three sections. The purpose of first part is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 8 macrolide antibiotics and lincosamides inside fish tissue, including erythromycin (ERM), oleandomycin (OLD), kitasamycin (KIT), tylosin (TYL), josamycin (JOS), spiramycin (SPM), tilmicosin (TIL), and lincomycin (LIN). Homogenized samples are first extracted with acetonitrile, dehydrated with sodium sulphate anhydrous, and then condensed. After the residue was redissolved in methanol and the extracts were partitioned with n-hexane to remove lipids, the sample is filterced and detected by LC/MS-MS using chromatography columns of Agilent HC-C18 (5£gm, 150 mm ¡Ñ4.6 mm). The mobile phase A was 5mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was acetonitrile. The analysis of 8 macrolide antibiotics and lincosamides can be achieved within 10 minutes with electrospray ionization-tandem mass spectrometry in positive mode using multiple reaction monitoring (MRM) for simultaneous detection.
The second part is to verify the method by regulation of European Union (EU) resolution scheme (2002/657/EC). In the case where the drug is set as allowed drug, the recovery rate under gradient addition according to MRL is between 93.64% to 106.67%, and the CV is between 0.27% to 7.17%. In the case where the drug is set as prohibited drug, the recovery rate under gradient addition according to MRPL is between 96.35%~104.88%, and the CV is between 6.77%~13.91%. As a result, the decision limit (CC£\) and the Detection capability (CC£]) of the 8 macrolide antibiotics and lincosamides is between 0.24 to 0.40£gg kg-1 and 0.33 to 0.49£gg kg-1.
The last section is to evaluate the stability of drugs in fish body under domestic preservation and process methods on fish, including refrigeration at -20¢J and cold storage at 4 ¢J. The test is implemented by adding the drug into fish tissue according to MRL and detecting the antibiotics residue after regulated 40 days. Besides, the effect on activity of drug residue in fish body after boiling at 100 ¢J is compared. The results show that the residual amount of spiramycin, josamycin, tilmicosin, and lincomycin is below 35% while that of erythromycin, oleandomycin, kitasamycin, and tylosin will be below 20%. Therefore, the drugs including erythromycin, josamycin, tylosin, and lincomycin will stay stably in fish tissue if they are stored under -20 ¢J. However, it may affect human health if the fish contains such antibiotic residues is not boiled.
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Investigation of the distribution of alkylphenol and alkylphenol polyethoxylates in main rivers and harbor areas of Kaohsiung city by LC-MS/MSChen, Jen-kun 04 September 2006 (has links)
Hou-Chin stream, Love river, and Chien-Chen river, the three main rivers in Kaohsiung city, flow through the populous residential and industrial areas. A large portion of sewage from domestic and industrial sources are discharged into these rivers, then the Love river and Chien-Chen river pour into the harbor area. In order to understand the pollution of alkylphenol polyethoxylates in these areas, water and sediment samples in Hou-Chin stream, Love river, Chien-Chen river and harbor area in Kaohsiung city were collected and the contents of alkylphenol and corresponding polyethoxylates were analyzed in this study.
LC/MS/MS was used as the analytical instrument which is relatively time-saving in comparison with other instruments. It is also more convenient due to the facts that no derivation or colorization are needed in sample pretreatment. The detection limit can reach to 0.03 ng/ml and recovery can be around 83.6~91.6%. It can analyze alkylphenols combinded with long ethoxylate chain with improved sensitivity and selectivity.
In the four sampling areas, the concentration of NPs in water were between 7.4~241.8ng/ml, and OPs were between 0.66~64.2ng/ml. The most contaminated water samples were found at Chih-Ping Bridge on the mainstream of Love river and Pau-Chu-Kou Dam Station and Min-Tsu Bridge on the tributary of Love river where the concentrations of NPs were greater than 200ng/ml, OPs were greater than 30ng/ml. We found that the main pollution sources were from Lung-Hsin Bridge, Tzu-Yu Bridge, Lung -Hua Bridge, and Pau-Chu-Kou Dam Station. The pollution sources of the Chien-Chen river were mainly from Chung-An Bridge and Chen-Chuan Bridge.
Concentration of NPs in upper sediments were between 633.1~2113.8ng/g, OPs were between 50.3~287.9ng/g. The highest concentration of NPs was at Ho-Ti Bridge on the mainstream of Love river, and the lowest concentration of NPs was at Chung-An Bridge on Chien-Chen river. The highest concentration of OPs was at Chen-Chuan Bridge in Chien-Chen river, and the lowest concentration of OPs was Min-Tsu Bridge on the tributary of Love river. The concentration of NPs in deeper sediments were between 523.9~1919.5ng/g, OPs were between 39.9~322.0ng/g. The highest concentration of NPs was at Chung-Hua Bridge on the tributary of Lover river, and the lowest concentration of NPs was at Chung-An Bridge on Chien-Chen river. The highest concentration of OPs was at Chi-Chin Fishing Port, and the lowest concentration of OPs was at Min-Tsu Bridge on the tributary of Love river.
The salinity of water samples and the total organic carbon in sediment sample will influence the distribution coefficient of alkylphenol polyethoxylates with different length of ethoxylate chains, their distribution coefficients were between 0.48~2.67.
In comparison with foreign studies, the concentrations of alkylphenol polyethoxylates of water and sediments amples in this study were between the highest and lowest values reported. However, the observed concentrations of alkylphenols in these study areas were higher then other rivers in Taiwan. These values were higher than the Probable No Effect Concentrations ( PNEC) of NP risk assessed by European Union. It can be concluded that the pollution of alkylphenol polyethoxylates of water and sediment is getting more serious in Hou-Chin stream, Love river, Chien-Chen river and harbor area in Kaohsiung city.
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The biochemical studies of peroxidase in Wasabia japonicaShieh, Chia-lin 12 February 2008 (has links)
The plant peroxidases (EC1.11.17) exit as a large family of isozymes. These isozymes have more than 50% amino acid sequence differences. The function of Wasaba japonica peroxides plays the role as IAA oxidases. The kinetics result shows Wasabia japonica peroxidases displayed affinity (Km = 17.1 £gM) for IAA. The kinetics results in Wasabia japonica peroxidases display affinity (Km = 80.6 £gM) for syringaldazine. LC/MS/MS technique described the data that has proven to be a method for identification and characterization of proteins. The soluble proteins extracted form Wasabia japonica was purified by gel filtration chromatography and two-dimensional gel electrophoresis (2-DE). LC-MS/MS analyses of 2-DE gel spots and identify proteins structure based on the protein fragmentation characteristics. The Mascot Search Results showed that Wasabia japonica peroxidase has a significant similarity (10%) with Arabidopsis thaliana peroxidase.
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Studies in Determination and Residues of Nitrofurans and Corresponding Metabolites by LC-MS/MS in TilapiaTsai, Chung-Wei 24 August 2009 (has links)
Nitrofurans have been widely used either in waterbath or feed additives for the prevention and treatment of aquatic products. The European Union was able to assign a maximum residue limit and prohibited nitrofurans used to animals in 1995, because of the potential carcinogenic effects of their residues on human health. This study is focusing on the analytical method of four kinds of commonly used nitrofurans and corresponding residual metabolites by LC-MS/MS. The detection limits of furazolidone, furaltadone, nitrofurazone and nitrofurntoin were 6.11, 3.63, 4.52 and 6.20 £gg kg-1,respectively. The detection limits of AOZ, AMOZ, SC and AH were 0.23, 0.30, 0.36, 0.53 £gg kg-1, respectively. The lightness is the main factor to cause the decomposition of nitrofurans. It is not significant for temperature to depredate nitrofurans. The adsorbtion of metabolites by the plastic tube was in the extraction procedure. Equipments in glass are suggested to be used for the sample pretreatment and plastic meterials are averted to be exercised.
About the comparation of determination of AOZ by ELISA and LC-MS/MS. The result demonstrated that the ELISA method might overestimate the residual AOZ content at low concentrations. The detection limit and recovery of the known addition were 0.05 £gg kg-1 and 108% for the LC-MS/MS method and 0.31 £gg kg-1 and 305% for the ELISA method, respectively.
The amounts of residual nitrofurans and metabolites in muscle, liver, gill and skin tissue of tilapia which were treated in different conditions were compared. The depletion data of bathing treatment group obtained showed similar be haviors of furazolidone, furaltadone, nitrofurazone, nitrofurantoin in tilapia which the residual time was less than 24 hr. The amounts of residual nitrofurans appeared the highest concentration in gill and the lowest concentration in muscle. Bonded residues of metabolites can be detected for at least 4 weeks after administration in muscle, skin, liver and gill. The concentrations of residual bonded metabolites were higher than non-bonded metabolites in gill and muscle besides liver during depletion periods. After bathing medication, there were more residual nitrofurans and corresponding metabolites in sea water tilapia than fresh water group, because sea water fish survives in high osmotic condition to reduce their urination. Nitrofurans and metabolites were deconstructed by enzyme in gills, livers, intestines and muscles. Then tissues of fish accumulated nitrofurans and metabolites soon after medication. The maturity of fish is one of facters to effect different residual concentration during depletion periods. Liver is the main tissue to deconstruct nitrofurans and metabolites for the bathing medication and intestine is the major tissue to decompose antibiotics for the feeding medicaton.
In this research, we built a completed way to determine nitrofurans and corresponding metatbolites. Comparation of fish in different conditions and different medicative ways were in this investigation. These results could be helpful for aquacultures and government institutions.
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Characterization of Protein Sumoylation in Response to Alkylation Stress in HEK 293 CellsManza, Linda Lee January 2007 (has links)
Stress conditions such as heat shock, UV, alkylating agents, and H2O2 have been shown to result in the modification of a variety of protein targets via the production of reactive electrophiles. These modifications can directly impact protein function or can alter posttranslational modifications, thus leading to a disruption of cellular regulatory processes. Recent studies have shown that stress-induced protein modifications can modulate posttranslational modification by the small ubiquitin related modifier (SUMO) family of proteins. Unlike ubiquitination, which primarily targets proteins for proteasomal degradation, sumoylation exerts a variety of effects including protein stabilization, subcellular localization, and the alteration of protein-protein interactions and transcriptional activity. To investigate the effects of alkylation and oxidative stress on sumoylation, HEK293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, Texas Red C5 bromoacetamide, hydrogen peroxide, and 4-hydroxynonenal (HNE), a highly reactive product of lipid peroxidation associated with oxidative stress. Western blot analysis revealed that the agents tested resulted in concentration-dependent changes in the patterns of SUMO-1 and SUMO-2/3 protein conjugation. Localization studies using western blot analysis and confocal immunofluorescence microscopy demonstrated that SUMO-1 protein conjugates were located primarily in the nucleus, whereas SUMO-2/3 protein conjugates were more equally distributed between the nucleus and the cytoplasm. SUMO-associated proteins were harvested from vehicle- and HNE-treated non-transfected HEK293 cells using agarose conjugated anti-SUMO-1 antibodies or from HA-SUMO-1- and HA-SUMO-3-expressing HEK293 cells using immunoaffinity chromatography. Multidimensional liquid chromatography-tandem mass spectrometry analyses resulted in the identification of 54 HA-SUMO-1-associated proteins and 37 HA-SUMO-3-associated proteins in vehicle-treated cells and 21 HA-SUMO-1- and HA-SUMO-3-associated proteins in HNE treated cells. Additionally, 27 SUMO-1-associated proteins were identified in the HNE-treated non-transfected cells. The functional classes of proteins targeted included RNA binding and processing proteins, metabolic enzymes, cytoskeletal regulators, and chaperone proteins. HNE treatment resulted in a near complete redistribution of both SUMO-1 and SUMO-3 to different targets. There was a 15% overlap in SUMO-1 and SUMO-3 associated proteins in vehicle-treated cells and a 10% overlap in HNE-treated cells indicating that SUMO proteins target distinct protein groups. These results indicate that protein modifying reactive electrophiles can regulate protein functions through the indirect alteration of endogenous posttranslational modifications.
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