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Aufgrund der hohen Zahl an Pflanzenschutzmittelwirkstoffen, die weltweit im Einsatz sind, wurden in der Rückstandsanalytik Multiscreening-Methoden etabliert. In diesem Zusammenhang findet die Flüssigkeitschromatographie (liquid chromatography (LC)) in Verbindung mit Quadrupol Time-of-Flight (Q-TOF)-Massenanalysatoren, welche sich durch eine hohe Auflösung und Massengenauigkeit auszeichnen, im Besonderen Anwendung. Eine Herausforderung sind und bleiben auch für diese Analysentechnik Matrixeffekte (ME), welche durch Verbindungen, die mit den Analyten co-eluieren, hervorgerufen werden und die quantitative und qualitative Analyse beeinträchtigen oder gar verhindern können. Die zweidimensionale LC (2D-LC) kann eine Möglichkeit darstellen, diese negativen Effekte durch eine verbesserte Matrixabtrennung zu minimieren. In der vorliegenden Arbeit wurde ein 2D-LC-System für den Einsatz als Multiscreening-Methode in der Rückstandsanalytik von Pflanzenschutzmittelwirkstoffen mittels Detektion am Q-TOF-Massenspektrometer getestet und optimiert. Anhand von ausgewählten Testanalyten, die einen weiten Säure- bzw. Basenstärke- und Polaritätsbereich abdeckten, wurde die Methode optimiert, sodass ein weiter Bereich zu erwartenden Analytverhaltens berücksichtigt wurde und der Analytumfang in der Folge ohne weitere Anpassungen der Methodenparameter (z. B. Ventilschaltzeiten) stark erweitert werden konnte. Darüber hinaus wurden die Ursachen für die Unterschiede im Elutionsverhalten einiger Analyten in der ersten LC-Dimension (Hydrophile Interaktionschromatographie (hydrophilic interaction chromatography (HILIC)) untersucht. Es wurde ein Berechnungsmodell postuliert, welches eine Abschätzung des Elutionsverhaltens von bisher nicht in der Methode implementierten Analyten ermöglicht. Die optimierte 2D-LC-Methode wurde schließlich einer auf der Quick Easy Cheap Effective Rugged Safe (QuEChERS)-Multimethode basierenden LC-TOF-Screening-Methode (QuE-LC-TOF-Methode) gegenübergestellt. Der Methodenvergleich erfolgte sowohl im positiven als auch im negativen Elektrosprayionisations-Modus (ESI-Modus). Er zeigte, dass die optimierte 2D-LC-TOF-Methode für den Screening-Einsatz unbekannter Proben auf Pflanzenschutzmittelrückstände geeignet ist und sogar eine bessere Detektierbarkeit der Analyten als die QuE-LC-TOF-Methode aufweist. Zudem wurde anhand von Matrix-Fingerprinting-Untersuchungen mit zehn Blankmatrices festgestellt, dass mit der QuE-LC-TOF-Methode generell eine größere Zahl an Verbindungen, insbesondere polarer Verbindungen mit Massen > 1000 Da, detektiert wird. Ein großer Teil dieser Verbindungen war auf den Einsatz der verwendeten Reagenzien (Extraktionssalze) zurückzuführen. Mittels der optimierten 2D-LC-TOF-Methode wurden viele dieser polaren Verbindungen abgetrennt und waren somit nicht mehr nachweisbar. Hinsichtlich der Ionisationspolarität zeigte sich, dass generell weniger Verbindungen im negativen ESI-Modus nachweisbar waren, unabhängig von der verwendeten Methode und der analysierten Matrix. Weiterhin wurden Untersuchungen zu ME mit beiden Methoden in beiden ESI-Modi durchgeführt. Dabei ließ sich mit Ausnahme für den polaren Elutionsbereich kein eindeutiger Zusammenhang zwischen der Anzahl der detektierten Matrixbestandteile und dem Ausmaß der ME feststellen. Die ME unterschieden sich in den beiden ESI-Modi vielmehr in ihrem Ausmaß (schwach/stark) als in ihrer „Richtung“ (Suppression/Erhöhung). Stärkere ME konnten überwiegend im positiven ESI-Modus beobachtet werden. Allerdings unterschieden sich bei Analyten mit ähnlichen Retentionszeiten die ME sowohl in ihrer Richtung als auch in ihrem Ausmaß. Die Ergebnisse deuten darauf hin, dass ME sowohl matrix- als auch substanzabhängig sind., A high number of active substances are used as pesticides worldwide. Consequently, multi-screening methods have been established for the analysis of pesticide residues. Especially liquid chromatography (LC) is frequently applied. Combined with quadrupole-time-of-flight (Q-TOF) mass analyzers, which are characterized by high resolution and mass accuracy, LC-high resolution mass spectrometry is a powerful tool for pesticide analysis. Matrix effects (ME) caused by compounds co-eluting with the analytes are a challenge for this analytical technique, because they can adversely affect or even prevent the quantitative or qualitative analysis. Two-dimensional LC (2D-LC) may be a way to minimize these negative effects through improved matrix separation. In the present work, a 2D-LC system coupled to a Q-TOF mass spectrometer was tested and optimized regarding its use for a multi-screening method in pesticide residue analysis. On the basis of selected test analytes covering a wide range of acid or base strength and polarity, the method was optimized in order to significantly extend the method scope without further adjustments to the method parameters (e.g., valve switching times). In addition, the reasons for the differing elution behavior of some analytes in the first dimension (hydrophilic interaction chromatography (HILIC)) were investigated. A calculation model was postulated that allows an estimation of the elution behavior of analytes that have not yet been implemented in the method. Finally, the optimized 2D-LC method was compared to an LC-Q-TOF screening method based on the Quick Easy Cheap Effective Rugged Safe (QuEChERS) multi-method (QuE-LC-TOF method). The method comparison was performed in both positive and negative electrospray ionization mode (ESI mode). It was shown that the optimized 2D-LC-TOF method was suitable for screening unknown samples for pesticide residues, and even had a better analyte detectability than the QuE-LC-TOF method. In addition, based on matrix fingerprinting studies with ten blank matrices, it was found that the QuE-LC-TOF method generally detected a larger number of compounds, especially polar compounds with masses >1,000 Da. Many of these compounds resulted from the usage of the reagents (extraction salts). By applying the optimized 2D-LC-TOF method, many of these polar compounds with high masses were separated and therefore no longer detectable. In addition, investigations on matrix effects were carried out with both methods and in both ESI modes. With the exception of the polar elution range, no clear correlation was found between the number of detected matrix components and the extent of the ME. Comparing both ESI modes, the ME differed much more in their extent (weak/strong) than in their direction (suppression/increase). Stronger ME could be observed predominantly in the positive ESI mode. However, analytes with similar retention times showed different ME, both in direction and extent. These results suggest that ME are both matrix- and compound-dependent.

Kontinuirano povećavanje upotrebe pesticida u suvremenoj poljoprivredi postaje neizbježno zbog učinkovitije proizvodnje hrane i samog porasta svjetske populacije. No, s druge strane sve je veća zabrinutost javnih institucija zbog štetnosti pesticida na ljude, druge organizme (domaće životinje, korisne insekte, pčele, ptice, ribe i dr.) i njihovu bio akumulaciju u samome tlu i svim vodenim ekosustavima. Stoga je u ovom radom analizirana fotolitička i fotokatalitička razgradnja s fotokatalizatorima titan dioksid (TiO2) i nanokompozitom titan doksida i ugljikovih nanocjevčica (TiO2/CNT) imidakloprida u pilot reaktoru s paraboličnim zrcalima (eng. compound parabolic collector, CPC). Prema kinetici razgradnje prvog reda, određene su približne konstante brzine fotolitičke razgradnje imidakloprida (k=0,0058 min-1) i fotokatalitičke razgradnje imidakloprida s TiO2 (k=0,0058 min-1) i s TiO2/CNT (k=0,00934 min-1). Primjenom TiO2/CNT fotokatalizatora može se postići brža razgradnja imidakloprida za 3,6 sati, u odnosu na fotokatalitičku razgradnju sTiO2. Analizom razgradbenih produkata, utvrđena su tri (m/z=212,0585; m/z=209,0589 te m/z= 211,0745) od četiri najčešća fotolitički razgradbena produkta imidakloprida, dok je svega jedan razgradbeni spoj (m/z= 274,0518) imidakloprida utvrđen pri fotokatalitičkoj razgradnji na TiO2 i TiO2/CNT fotokatalizatorima. To ukazuje na potrebu detaljnije analize, budući da su se eksperimenti provodili pri realnim uvjetima sunčeva zračenja. The continuous increase in the usage of pesticides in modern agriculture is becoming inevitable due to more efficient food production and the increase in world population. On the other hand, harmful effects of pesticides on humans and other organisms (domestic animals, beneficial insects, bees, birds, fish, etc.) cause concerns of public institutions due to their bio accumulation effect on the soil and aquatic ecosystems. Therefore, in this paper, the photolytic and photocatalytic degradation with photocatalysts titanium dioxide (TiO2) and nanocomposite of titanium dioxide and carbon nanotubes (TiO2/CNT) of imidacloprid in a pilot reactor with compound parabolic mirrors (CPC) was analysed. According to the kinetics of the pseudo first-order degradation, the apparent rate constants of the photolytic degradation of imidacloprid (k=0.0058 min-1) and the photocatalytic degradation of imidacloprid with TiO2 (k=0.0058 min-1) and with TiO2/CNT (k=0.00934 min-1) were determined. Faster degradation (3.6 hours) of imidacloprid can be achieved using TiO2/CNT photocatalyst in comparison to the usage of TiO2. The analysis of the degradation products revealed three (m/z=212.0585; m/z=209.0589 and m/z= 211.0745) out of the four most common photolytic degradation by-products of imidacloprid, while only one degradation compound (m/z = 274.0518) of imidacloprid was determined during photocatalytic degradation on TiO2 and TiO2/CNT photocatalysts. It indicates the need for a more detailed analysis since the experiments were carried out under the actual conditions of solar radiation.

Mikroonečišćivala su biološka ili kemijska onečišćivala koja ulaze u podzemne i površinske vode u tragovima kao rezultat ljudskih aktivnosti. Ti onečišćivači uključuju bezbroj prirodnih i sintetskih organskih spojeva, od kojih su mnogi označeni kao "nastali" uglavnom zbog toga što se tradicionalno ne nadziru ili ne reguliraju u uzorcima okoliša. Zbog njihovog negativnog utjecaja na okoliš i činjenice da uzrokuju zdravstvene, ekonomske i ekološke probleme, postali su pitanje od nacionalnog i globalnog značaja. Cilj ovog rada je analiza uzoraka s tri lokacije na području sjeverozapadne Hrvatske, kako bi se utvrdila prisutnost mikroonečišćivala poput farmaceutika, hormona, antibiotika i pesticida. Metoda koja je korištena za ispitivanje uzoraka u ovom radu je masena spektrometrija i tekuća kromatografija na uređaju Agilent 6350 Q-TOF LC/MS. Micro pollutants are biological or chemical pollutants that enter groundwater and surface waters as trace pollutants as a result of human activites These pollutants include a multitude of natural and synthetic organic compounds, many of which are labelled as “formed”, mainly because they are not traditionally controlled or regulated in environmental samples. Due to their negative impact on the enviroment and the fact that they cause health, economic and environmental problems, they have become an issue of national and global importance. The aim of this master thesis is to analyse samples from three locations in the area of north-western Croatia, in order to determine the presence of micro pollutants such as pharmaceuticals, hormones, antibiotics and pesticides. The method used to test the samples is mass spectrometry and liquid chromatography on an Agilent 6350 Q-TOF LC / MS.

Microalgae and their potential for food supplementation are a growing trend in modern society. Whole microalgal biomass and their extracts are rich in nutrients which give various health benefits. To further improve their characteristics as food supplements a co-cultivation with probiotic strains emerged as a new innovative way to increase nutrititional value. In order to increase probiotic strain acitivites, algae are usually added in powder form to cultivating broth. In this study, we performed co-cultivation of probiotic bacteria and viable microalga Chlorella vulgaris. Our main goal was to investigate if this approach can benefit to both organisms. We focused on optimization of growth conditions, examing inoculation ratio, temperatures, culturing volumes and media in correlation with biomass accumulation. Determination of C. vulgaris biomass composition has been performed using Nile Red (NR) method with flourescence microscopy for lipid accumulation ; spectrophotometry for pigments and Lowry method for protein yield. Metabolite products synthesized and released in culture medium, as well as metabolites embedded in biomass composition, were analysed using quadrupole time-of-flight (q-TOF) mass spectrometry revealing production of new compounds. Results of these comprehensive analysis will be presented.

In the face of mounting global antibiotic resistance, which has become a critical clinical problem, antimicrobial peptides (AMPs) have received considerable interest as new therapeutics with the efficacy for the treatment of multidrug-resistant (MDR) infections due to their novel mechanism. However, certain inherent shortcomings such as instability seriously limit their systemic applications in the clinic. In this study, we intend to clarify the connection between three configurations of IK8 and their stability in plasma and liver S9 of various species by confirming the metabolites. The structural information of these metabolites was scanned and identified using HPLC and Q-TOF, respectively. The results found that IK8-D exhibits superior stability, compared with IK8-2D and IK8-L in plasma and liver S9 incubation, which indicated D- type amino acids could significantly increase the stability of antimicrobial peptides.

In this study, nine forced degradation products of maraviroc were found using chemometric analysis. This antiretroviral drug was subjected to photolytic, oxidative, as well as neutral, basic and acidic hydrolysis stress conditions. Additionally, its electrochemical transformation on platinum, gold and glassy carbon screen-printed electrodes was examined. This study showed that maraviroc is especially susceptible to UVA, H2O2 and electrochemical degradation, while being resistant to neutral and acidic hydrolysis. A cluster analysis showed that the electrochemical transformation, with particular reference to the platinum electrode, is able to partially simulate the forced degradation processes, especially in the context of redox reactions. These findings indicate that the electrochemical methods can be considered as quick and relatively low-cost supplements to the commonly applied forced degradation procedures.

Background Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography. Results Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79–107%. Conclusion The results show the reliability of combined reversed phase liquid chromatography–tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species (Seriola fasciata) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands (Portugal). European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03 Xunta de Galicia | Ref. ED481A-2018/207

Tese de mestrado em Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-05-07T15:29:36Z No. of bitstreams: 1 ulfc126209_tm_Margarida_Isidro.pdf: 3456296 bytes, checksum: 63c2efdf9ba6e9deed22a10c31456c50 (MD5) Made available in DSpace on 2021-05-07T15:30:12Z (GMT). No. of bitstreams: 1 ulfc126209_tm_Margarida_Isidro.pdf: 3456296 bytes, checksum: 63c2efdf9ba6e9deed22a10c31456c50 (MD5) Previous issue date: 2020

ST segment elevation myocardial infarction (STEMI) is one of the most common global causes of cardiovascular disease-related death. Several metabolites may change during STEMI. Hence, analysis of metabolites in body fluid may be considered as a rapid and accurate test for initial diagnosis. This study has therefore attempted to determine the variation in metabolites identified in the serum of STEMI patients (n = 20) and 15 controls. Samples collected from the Cardiology Department, Medical Faculty, Ataturk University, were extracted by liquid-liquid extraction and analysed using liquid chromatography quadrupole time-of-flight mass spectrometry. The METLIN database was used for the identification and characterization of metabolites. According to Q-TOF/MS measurements, 231 m/z values, which were significantly different between groups (P < 0.01 and fold analysis >1.5) were detected. Metabolite identification was achieved via the Human Metabolome database. According to the multivariate data analysis, leucine, isoleucine, l-proline, l-alanine, glycine, fumaric acid, citrate, succinate and carnitine levels were decreased, whereas levels of propionic acid, maleic acid, butyric acid, urea, oleic acid, palmitic acid, lysoPC [18:2(9Z)], glycerol, phoshpatidylethanolamine, caffeine and l-lactic acid were increased in STEMI patients compared with controls. In conclusion, malonic acid, maleic acid, fumaric acid and palmitic acid can be used as biomarkers for early risk stratification of patients with STEMI. C1 [Gundogdu, Gulsah] Pamukkale Univ, Dept Physiol, Fac Med, TR-20070 Denizli, Turkey. [Senol, Onur; Demirkaya Miloglu, Fatma] Ataturk Univ, Dept Analyt Chem, Fac Pharm, Erzurum, Turkey. [Koza, Yavuzer; Gundogdu, Fuat] Ataturk Univ, Dept Cardiol, Fac Med, Erzurum, Turkey. [Hacimuftuoglu, Ahmet; Abd El-Aty, A. M.] Ataturk Univ, Fac Med, Dept Med Pharmacol, Erzurum, Turkey.

Baby bottles made of polypropylene, Tritan® and silicone were evaluated regarding the migration of non-volatile compounds using UPLC-QTOF-MS. Twenty-seven compounds were identified. In all polypropylene samples the migration of 2.2′-(tridecylimino)bis-ethanol and derivatives thereof were detected in concentrations below the specific migration limit (1.2 mg.kg−1). Furthermore, clarifying agents and glycerol derivatives were detected. Tritan baby bottle showed the migration of one slip additive. On the other hand, twenty compounds were detected in silicone baby bottles. Most of them were unknown compounds derived from acrylates. Once the migrants were identified, the risk assessment was carried out using the Threshold of Toxicological Concern (TTC) approach. The risk assessment of migrants coming from silicone samples showed levels above the threshold recommended as safe for babies.

Normal 0 21 false false false PL X-NONE X-NONE Background: The radiation sterilization is one of the best methods for sterilizing vulnerable for degradation drugs like cefozopran hydrochloride. Results: Chemical stability of radiosterylized cefozopran hydrochloride, was confirmed by spectrophotometric and chromatographic methods. EPR studies showed that radiation has created some radical defects which concentration was no more than several dozen ppm. The antibacterial activity of cefozopran hydrochloride irradiated with a dose of 25 kGy was unaltered for Gram-positive bacteria but changed for two Gram-negative strains. The radiation sterilized cefozopran hydrochloride was not in vitro cytotoxic against human CCD39Lu normal lung fibroblast cell line. Conclusions: Cefozopran hydrochloride in solid state is not resistant to radiation sterilization and this method cannot be used for sterilization of this compound. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif"; mso-ansi-language:PL; mso-fareast-language:PL;}

The influence of ionising radiation on the physicochemical properties of meropenem trihydrate in solid state was studied for doses of e-beam radiation: 25 kGy and 400 kGy. In the first part of our studies, we evaluated the possibility of applying radiosterilization to obtain sterile meropenem. No changes for meropenem irradiated with a dose of 25 kGy, the dose required to attain sterility, was confirmed in the results of spectroscopic (FT-IR), thermal (DSC, TGA) and X-ray powder diffraction (XRPD) studies. The radiation dose of 25 kGy produces no more than about 1500 ppm of radical defects. The chromatographic studies of irradiated meropenem in solutions did not show any chemical degradation. Moreover, the antimicrobial activity of meropenem irradiated with the dose of 25 kGy was unchanged. Based on the received results, we can conclude that radiostelization is a promising, alternative method for obtaining sterile meropenem. In the second part of the research, meropenem was exposed to e-beam radiation at the 400 kGy dose rate. It was confirmed, that reducing of antimicrobial activity could be connected with the degradation of &beta, lactam ring and changes in the trans-hydroxyethyl group. Apart from chemical changes, changes in the physical stability of irradiated meropenem (400 kGy) was also observed.

Ultra high performance liquid chromatography (UHPLC), coupled with accurate quadrupole-time-of-flight (Q-TOF) mass spectrometry, was used for the stability study of acetylsalicylic acid within a variety of different organic solutions: methanol, ethanol, propan-2-ol, acetonitrile, tetrahydrofuran and 1,4-dioxane. With the use of gradient elution chromatography and mass spectrometry detection in negative ionization, MS and MS/MS spectra were recorded simultaneously. In addition, quantitative, as well as qualitative analysis was performed during one assay. The stability of acetylsalicylic acid in such solutions was tested at room temperature, in a 12h period. In the work, in all cases, only one main degradation product, salicylic acid, was found. What is more, the work revealed that the degradation of aspirin in the tested organic solutions yields apparent second-order kinetics. The study also demonstrated that acetonitrile and 1,4-dioxane turned out to be the most stable solvents, and an above 80% of initial concentration of acetylsalicylic acid was found in this case. Furthermore, the most popular analytical solvents, methanol and ethanol, were found to be very unstable media. Herein, below 40% of initial concentration of acetylsalicylic acid was seen after 12h. The obtained results were also compared with the degradation of acetylsalicylic acid in a water solution. In this situation, only about 25% of the analyzed compound was resolved to salicylic acid in the same time frame.

Neadekvatna uporaba antibiotika u humanoj i veterinarskoj medicini dovodi do razvoja antimikrobne rezistencije, širenja otpornosti mikroorganizma te pojave njihovih ostataka u namirnicama životinjskog porijekla. Jedna od najčešće primijenjenih vrsta antibiotika su cefalosporini. Da bi ih adekvatno identificirali i kvantificirali bilo je potrebno razviti metodu i ispitati utjecaj metode za identifikaciju rezidua cefalosporina u mišićima goveda, svinje, peradi i ribe primjenom tekućinske kromatografije ultravisoke djelotvornosti sa spektrometrijom masa i analizatorom vremena preleta (UHPLC/Q-TOF-MS), što je ujedno i glavni cilj ovog rada. Također, validirana metoda za određivanje cefalosporina u mišićima različitih životinjskih vrsta visoke je specifičnosti, točnosti i preciznosti te zadovoljava kriterije o provođenju analitičkih metoda i tumačenju rezultata prema važećoj Uredbi komisije 2002/657/EZ. Metoda je primijenjena u rutinskoj analizi na Hrvatskom veterinarskom institutu. Na prisustvo 9 najčešćih cefalosporina testirana su 42 uzorka pri čemu nije pronađen ni jedan pozitivan uzorak. Inadequate use of antibiotics in human and veterinary medicine leads to the development of antimicrobial resistance, the spread of microorganism resistance and the appearance of their residues in food of animal origin. One of the most frequently used types of antibiotics are cephalosporins. In order to identify and quantify, it was necessary to develop a method and to examine the effect of the method for identifying the residual cephalosporin residues in cattle, pigs, poultry and fish muscles using ultra-high performance liquid chromatography and high resolution mass spectrometry (UHPLC/Q-TOF-MS), which is also the main objective of this paper. Validated method for the determination of cephalosporin in the muscles of different animal species is of high specificity, accuracy and precision and meets the criteria for the conduct of analytical methods and interpretation of results under the current Commission Regulation 2002/657 / EC. The method was applied in a routine analysis at the Croatian Veterinary Institute. In the presence of 9 most common cephalosporins, 42 samples were tested and no positive samples were found

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two-dimensional gel electrophoresis and separation by ion-exchange and reverse-phase high-performance liquid chromatography followed by mass spectrometry using tanden matrix-assisted laser desorption/ionization with time-of-flight (MALDI-TOF/TOF) mass spectrometry and electrospray ionization-quadrupole with time-of-flight (ESI-Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10-ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.

International audience; Polymeric tannins from grapes have always been reported as an unresolved broad peak in HPLC chromatograms, and this has severely limited their identification to date. This study aimed to disassemble this broad peak and explore the polymeric tannin molecules inside. By applying centrifugal partition chromatography (CPC), an efficient separation approach was developed to split the broad peak of grape seed tannins into fractions. Then, the fractions were analyzed by Q-ToF (quadrupole time-of-flight mass spectrometry) to determine the corresponding structures of the tannins. The results suggest that grape seed polymeric tannins were eluted consecutively according to their degree of polymerization (DP). Condensed tannins identified in wine grape seed have a range of DP and degree of galloylation (DG) up to 20 and 11, respectively. The molecular mass of the largest molecule detected was 6067. To our knowledge, this is the first report to offer an insight into the broad peak of polymeric tannins found with HPLC and to characterize the tannins with a DP up to 20 as shown by HRMS and MS/MS data.

Campylobacter jejuni is the major cause of bacterial gastroenteritis in man, while it is generally regarded as a commensal of the avian gut. Consumption and handling of contaminated poultry meat products are major risk factors for human infection. The body temperature in man (37 °C) and chickens (42 °C) differ markedly, and differential gene regulation and protein expression at different temperatures may in part explain the behaviour in the two hosts. We performed proteomics analyses with C. jejuni cells grown at 37 °C and 42 °C. Time-of-flight mass spectrometry (Q-Tof) analysis was carried out after samples were digested with the Filter-Aided Sample Preparation (FASP) method and peptides were fractionated by strong anion exchanges. Differentially regulated proteins were identified by Mascot and Scaffold analyses. Triple quadrupole (QQQ) mass spectrometer analysis confirmed that a total of 33 proteins were differentially regulated between 37 °C and 42 °C. Several upregulated proteins were selected for their corresponding gene knock-out mutants to be tested for their virulence in the Galleria mellonella model. To correlate with other tissue/animal models, the GADH mutant was selected for its reduced ability to colonize chickens. At 37 °C, the mutants of outer membrane protein Omp50 and Chaperone GroEL significantly increased virulence; while at 42 °C, the mutants of YceI, Omp50, and GADH reduced virulence against Galleria mellonella compared with the wild type strains. The results of current and previous studies indicate that GADH is a virulent factor in G. mellonella and a colonization factor in chickens. The workflow of this study may prove a new way to identify stress related virulent factors. The implications of these findings are discussed for pathogenesis in the model and other hosts.

Background Toddalia asiatica root bark as an effective hemostatic natural medicine or Chinese materia medica was applied in China for long history, its complex drug action mechanisms and unclear substance basis have been constraining the development of this drug. Results An intelligentized strategy by LC-ESI Q-TOF MSE was presented in this study for rapid identification of hemostatic chemical constituents from this natural medicine. Chromatographic separation was performed on a C18 column (150 mm × 2.1 mm, 1.8 μm), the MSE data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. To reduce the false positive identifications, structural confirmation was conducted by comparison with the isolated reference standards (tR and MS, MS/MS data) or matching with natural product databases. Bioassay-guided fractionation of the extract of T. asiatica root bark was also carried out. Conclusions As a consequence, 31 natural compounds in T. asiatica root bark got putatively characterized. There were four main coumarins, isopimpinellin (Cp.23), pimpinellin (Cp.24), coumurrayin (Cp.30) and phellopterin (Cp.34) isolated and identified from T. asiatica root bark. The present study provided candidate strategy that helps to effectively identify the primary natural compounds of TCM or other complex natural medicines, and then promote development and application of natural medicines and their medicinal resources.

Background: Coptis chinensis Franch is a traditional Chinese medical herb. Objective: In this article, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to rapidly, qualitatively, and comprehensively identify the components in Coptis chinensis Franch. Materials and Methods: Chromatographic separation was achieved on an Agilent Zorbax RRHD Eclipse Plus C18 column. The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) with a gradient program. Qualitative analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer, which was equipped with a Dual AJS ESI source operating in negative mode. Results: A total of 30 alkaloid and non-alkaloid components of Coptis chinensis Franch were identified in only 14 min. Conclusion: This study helped to provide a basis for the quality control of Coptis chinensis Franch. SUMMARY Qualitative analysis method of chlorogenic alkaloids and non-alkaloids in Coptis chinensis Franch is developed by Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method.Established UPLC-Q-TOF-MS/MS analysis method is validated with rapidness and accuracy.The developed method was successfully applied for qualitative analysis of Coptis chinensis Franch sample collected from cultivation place in China. Abbreviations used: Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. pos: positive, neg: negative. neg: negative.

For the first time, the influence of ionising radiation on the physicochemical properties of ertapenem in solid state was studied. During our studies, we evaluated the possibility of applying radiosterilization to obtain sterile ertapenem. Spectroscopic (Fourier Transform Infrared (FT-IR)), thermal (differential scanning calorimetry (DSC), chromatography (High-Performance Liquid Chromatography (HPLC) and HPLC-MS), and X-ray powder diffraction (XRPD) studies shown that irradiation of ertapenem with the 25 kGy, the dose required to achieve sterility, does not change the physicochemical properties of the studied compound. The antimicrobial activity of ertapenem irradiated with the dose of 25 kGy was only reduced for one species. Based on the received results, we can conclude that radiostelization is a promising alternative method of obtaining sterile ertapenem. In our studies, ertapenem was also exposed to e-beam radiation with a dose of 400 kGy. It was determined that two novel degradation products that are structurally differently to degradants formed during hydrolysis and thermolysis.

The conditioning effect in metabolomic studies is the phenomenon of initial variation of analytical results in the first 5-10 injections of a biological sample in chromatographic systems. The deviation manifests itself as a drift in retention time, peak area and in multivariate analysis. It is a major quality assurance problem in the metabolomic field and if not accounted for would result in high analytical variance. The aim of this study was to investigate the conditioning effect and to gain further knowledge about it. The study was carried out on UPLC of hydrophilic liquid chromatography (HILIC) type coupled to quadrupole time of flight (QTOF) MS. A systematic study was designed to investigate the effects of the age of the analytical column. An investigation into certain matrix components as a possible cause of the conditioning effect was made. Different sample preparation methods were investigated. One result showed that no conditioning could be seen and the system appeared stable from the first injection. Differences in sample composition between samples with conditioning effect and samples without conditioning effect were investigated. No correlation between conditioning effect and levels of certain matrix compounds could be found. More studies of correlation between sample composition and the amount of conditioning occurring is needed. Some samples appear to have no retention time drift but have a significant drift in peak area and in multivariate analysis. This is an indication that the conditioning effect should be analysed in more ways than one before determining if a system is stable.

Until now, most metabolomics protocols have been optimized towards high sample throughput and high metabolite coverage, parameters considered to be highly important for identifying influenced biological pathways and to generate as many potential biomarkers as possible. From an analytical point of view this can be troubling, as neither sample throughput nor the number of signals relates to actual quality of the detected signals/metabolites. However, a method’s selectivity for a specific signal/metabolite is often closely associated to the quality of that signal, yet this is a parameter often neglected in metabolomics. This thesis demonstrates the importance of considering selectivity when developing NMR and LC-MS metabolomics methods, and introduces a novel approach for measuring chromatographic and signal selectivity in LC-MS metabolomics. Selectivity for various sample preparations and HILIC stationary phases was compared. The choice of sample preparation affected the selectivity in both NMR and LC-MS. For the stationary phases, selectivity differences related primarily to retention differences of unwanted matrix components, e.g. inorganic salts or glycerophospholipids. Metabolites co-eluting with these matrix components often showed an incorrect quantitative signal, due to an influenced ionization efficiency and/or adduct formation. A novel approach for measuring selectivity in LC-MS metabolomics has been introduced. By dividing the intensity of each feature (a unique mass at a specific retention time) with the total intensity of the co-eluting features, a ratio representing the combined chromatographic (amount of co-elution) and signal (e.g. in-source fragmentation) selectivity is acquired. The calculated co-feature ratios have successfully been used to compare the selectivity of sample preparations and HILIC stationary phases. In conclusion, standard approaches in metabolomics research might be unwise, as each metabolomics investigation is often unique. The methods used should be adapted for the research question at hand, primarily based on any key metabolites, as well as the type of sample to be analyzed. Increased selectivity, through proper choice of analytical methods, may reduce the risks of matrix-associated effects and thereby reduce the false positive and false negative discovery rate of any metabolomics investigation.

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.

Targeted proteomics for biomarker verification/validation is a challenge for MS instrumentation as it requires maintaining the sensitivity and dynamic range over a large number of samples to precisely quantify specific subsets of peptides from complex proteomes. These verification/validation tasks have traditionally been performed on triple quadrupole instruments, using multiple (or selected) reaction monitoring (MRM or SRM) with stable isotope-labeled standards (SIS). In this study, we investigate the efficiency of an ultra-high resolution benchtop Q-TOF platform, typically used for discovery approaches, for targeted quantitation of peptides in a plasma tryptic digest. Two different quantification methods were investigated enabling quantification at either the precursor (HR-XIC) or fragment (DIA) ion level.

Photodegradation of quetiapine under UVC irradiation in methanol solution was investigated and structural elucidation of its photodegradation products was performed with the use of the reversed phase UHPLC system coupled with accurate mass hybrid ESI-Q-TOF mass spectrometer. During one run all essential data for the determination of photodegradation kinetics and for the structural elucidation of the products was collected with the use of auto MS/MS mode. Five degradation products were found and their masses and formulas were obtained with high accuracy (0.26–5.02 ppm). For all the analyzed compounds, MS/MS fragmentation spectra were also obtained allowing structural elucidation of the unknown degradation products and indicating photodegradation pathways of quetiapine. The main photodegradation product was identified as 2-[2-[4-(5-oxidodibenzo[b,f][1,4]thiazepin-11-yl)-1-piperazinyl]ethoxy]-ethanol and the photodegradation reaction yields the first-order kinetics with the rate constant k = 0.1094 h−1.

Condensed tannins and polymeric pigments are two families of macromolecules which play important roles on the organoleptic quality of red wine. Their oligo-polymeric structures and their evolutions during red wine aging are poorly known. The objective of this study is to deepen our knowledge of their structures and their evolutions during red wine aging. During our work, we characterized, for the first time in plant kingdom, a new family of condensed tannins named : crown condensed tannins, which have specific properties. They are more polar than B-type tannins. During red wine aging, the concentration of B-type tannins decreased while the crown tannins remained stable. Their concentrations are higher in Syrah wines than in wines made from Cabernet Sauvignon and Merlot grapes. From a sensory point of view, a strong correlation between the concentration of crown tannins and the intensity of astringency rated by the tasters was obtained. The first biologic activity assay of the crown tetramer showed an interesting inhibitory activity on aggregation of β-amyloid peptide involved in Alzheimer's disease. At the same time, a new quantification method of ethylidene bridge linked condensed tannins via a specific marker using fluorescence detection has been developed. Concerning polymeric pigments with different types of inter-flavonoids linkages, their acidic depolymerization mechanisms have been clarified for the first time and a quantification method using a UPLC-Q-TOF system has been developed. Oligo-/polymeric pigments contributed more to the color of old wine than mono-/dimeric pigments. The concentration of the pigmented oligo-polymers with A type linkages is more stable than that of polymeric pigments by B-type linkages or by ethylidene bridge during red wine aging. In addition, during our study of polymeric pigments, A-F(A type)-F(n) and F(n)-A-F(A type) structures as well as the acetylated form of trimeric anthocyanin and acetylated forms of dimeric anthocyanin linked by ethylidene bridge have been shown for the first time.; Les tanins condensés et les pigments polymérisés sont deux grandes familles de macromolécules qui jouent des rôles importants sur la qualité organoleptique du vin rouge. Leurs structures oligo-polymériques ainsi que leurs évolutions durant le vieillissement du vin rouge sont mal connues. L’objectif de cette étude est d’approfondir notre connaissance sur leurs structures et leurs évolutions durant le vieillissement. Durant ce travail, nous avons caractérisé, pour la première fois dans le règne végétal, une nouvelle sous-famille de tanins condensés nommés : les tanins condensés couronnes, qui ont des propriétés spécifiques. Ils sont plus polaires que des tanins condensés de type B. Durant le vieillissement, les concentrations des tanins de type B diminuent alors que celles des tanins couronnes restent plutôt stables. Leurs concentrations sont plus élevées dans les vins issus de cépage Syrah que dans les vins issus de cépage Cabernet Sauvignon et Merlot. Au niveau sensoriel, une forte corrélation entre la concentration des tanins couronnes et l’intensité d’astringence ressentie par les dégustateurs a été montrée. Un premier test d’activité biologique du tétramère couronne montre une activité inhibitrice intéressante de l’agrégation du peptide β-amyloïde impliquée dans la maladie d’Alzheimer. En même temps, une nouvelle méthode de quantification des tanins condensés liés par pont éthylidène via un marqueur spécifique en utilisant une détection par fluorescence a été développée. Concernant les pigments polymérisés avec différents types de liaisons inter-flavonoïdes, leurs mécanismes de dépolymérisation chimique en milieu acide ont été clarifiés et une méthode de quantification sur un système UPLC-Q-TOF a été développée. Les pigments oligo-/polymériques contribuent plus à la couleur du vin vieux que les pigments mono-/dimériques. La concentration des structures oligo-polymériques ayant des liaisons de type A est plus stable que celle des pigments polymérisés par des liaisons de type B ou par le pont éthylidène durant le vieillissement du vin. De plus, durant nos investigations sur les pigments polymérisés, les structures de type A-F(type A)-F(n) et de type F(n)-A-F(type A) ainsi que les formes acétylées d’anthocyane trimère et les formes acétylées des anthocyanes dimères liées par pont éthylidène ont été montrées pour la première fois.

Nowadays, chromatographic methods coupled with mass spectrometry are the most commonly used tools in metabolomics studies. These methods are currently being developed and various techniques and strategies are proposed for the profiling analysis of biological samples. However, the most important thing used to maximize the number of entities in the recorded profiles is the optimization of sample preparation procedure and the data acquisition method. Therefore, ultra high performance liquid chromatography coupled with accurate quadrupoletime- of-flight (Q-TOF) mass spectrometry was used for the comparison of urine metabolomic profiles obtained by the use of various spectral data acquisition methods. The most often used method of registration of metabolomics data acquisition – TOF (MS) was compared with the fast polarity switching MS and auto MS/MS methods with the use of multivariate chemometric analysis (PCA). In all the cases both ionization mode (positive and negative) were studied and the number of the identified compounds was compared. Additionally, various urine sample preparation procedures were tested and it was found that the addition of organic solvents to the sample noticeably reduces the number of entities in the registered profiles. It was also noticed that the auto MS/MS method is the least efficient way to register metabolomic profiles.

The aim of this master thesis project was to develop methods for sample preparation and analysis by LC-MS suitable for global metabolomics of human plasma samples. In this thesis six different methods was tested, based on precipitation, solid phase extraction (SPE), and ultrafiltration. The methods differ in their mechanisms of action, but the end goal is the same, to remove the proteins and other high molecular weight compounds from the samples and to retain as many metabolites as possible in a reproducible manner. The LC-MS analysis were performed on a C18 and a HILIC type column using electrospray ionization (ESI) with both positive and negative ionization to cover as much of the metabolome as possible. The MarkerLynx software was then used to extract features from the chromatograms. The coefficient of variation (CV) was calculated and the features with CV > 30 % were removed. The features were then used to compare the methods with each other in order to see whether any of the methods yielded the same capture of features. The largest amount of features was detected for precipitation with MeOH when using a C18 type column. For HILIC type columns precipitation with MeOH with a small addition of H3PO4 resulted in most unique features detected. The ionization mode was found to be less important, compared whit the choice of column, but more features was detected using positive mode than negative mode.

Ziele dieses mehrmonatigen Projekts waren die Erstellung einer hochauflösenden Spektrenbibliothek mittels Quatrupol Time-of-Flight Massenspektrometrie (Q-TOF-MS), - gekoppelt mit einer Ultra high pressure liquid chromatography (UHPLC) - für illegale Arzneimittel mit den Parametern Retentionszeit, Lösungsmittel, MS und MS² Spektrum. Die erhaltenen Spektren der Substanzen wurden mit den entsprechenden vorhandenen MS²-Spektren der In-house Ionenfalle MS Spektrenbibliothek verglichen, um eine Weiterverwendung dieser Spektren zu beurteilen. Zusätzlich wurden die Nachweisgrenzen aller Substanzen bestimmt, um die Anwesenheit in pharmakologisch aktiver Menge in den zu untersuchenden Proben weitestgehend ausschließen zu können. Es wurde eine UHPLC-Q-TOF Methode etabliert, die sowohl eine Trennung, als auch eine Abtrennung von Matrix ermöglicht unter der Berücksichtigung von Totzeit (die Matrix soll nicht retendiert werden, und nicht mit der Totzeit eluiert werden; Vermeidung der irreversiblen Adsorption der Substanzen auf der Säule), Ionisierung und Zerfall. Durch die erstellte hochauflösende Spektrenbibliothek wurde die Identifizierung von Wirkstoffen in illegalen Arzneimitteln erheblich beschleunigt. Objectives of this several months lasting project consisted of creating a high-resolution spectral library using quadrupole time-of-flight mass spectrometry (Q-TOF-MS)-coupled with a ultra high pressure liquid chromatography (UHPLC) for illegal drugs with the parameters retention time, solvents, MS and MS² spectrum. The resulting spectra of the compounds were compared with the corresponding existing MS² spectra of the in-house ion-trap-MS spectra library to assess further use of these spectra. In addition, the detection limits of all substances were determined to rule out the presence largely in pharmacologically active amount (in examined samples). A UHPLC-Q-TOF method was established containing both, a resolution and the separation of matrix that allows consideration of dead time ( Matrix should not be retained and not eluted with the dead time; avoid the "sticking" of the substances on the column), ionization and decay. By the created high-resolution spectral library the time for the identification of active compounds in illegal drugs has been greatly decreased. vorgelegt von: Alexander Gaco Masterarbeit Wien, FH Campus Wien 2014

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014 Submitted by Repositório FFUL (repositorio@ff.ul.pt) on 2017-11-07T12:20:52Z No. of bitstreams: 1 MICF_Marta_Rico.pdf: 4356382 bytes, checksum: eef0a38d046dc055a150c9bbb32f6a61 (MD5) Made available in DSpace on 2017-11-07T12:21:08Z (GMT). No. of bitstreams: 1 MICF_Marta_Rico.pdf: 4356382 bytes, checksum: eef0a38d046dc055a150c9bbb32f6a61 (MD5) Previous issue date: 2014 University of Eastern Finland (Kuopio).

The development of analytical tools for organic geochemistry analysis has increased these past years. This development has allowed answering many questions about organic matter composition. However, many issues remain to be clarified including the characterization of high molecular weight fractions and monitoring the reactivity of organic matter. This thesis has focused on both (i) existing method improvements for fossil organic geochemistry analysis but also on (ii) developing a new type of coupling between the size exclusion chromatography (SEC) and the APPI-QTOF mass spectrometry for high molecular weight weakly polar fractions. Adjustments on APPI-QTOF mass spectrometry have allowed a better understanding of polyaromatic organic contaminant reactivity in presence of mineral matrices. The success of this coupling has allowed a better understanding of the structure of asphaltenes. However despite the "simplification" obtained by the SEC, the large amount of information remains difficult to interpret and time-consuming. A mathematical model has been developed based on numerical and statistical analysis of mass spectra, allowing direct comparison of mass spectra and being able to identify several types of information such as origins of samples, monitoring of physico-chemical processes and also the efficiency of soil recovery treatments as well as the identification of analytical protocols.; Le développement des outils analytiques pour l'analyse de la matière organique complexe en géochimie organique a connu de nombreuses avancées ces dernières années. Ce développement a permis de répondre à un grand nombre de questions quant à la composition de la matière organique. Cependant, beaucoup des points restent encore à élucider comme notamment la caractérisation des fractions de hauts poids moléculaires ainsi que le suivi de la réactivité de la matière organique. Ce travail de thèse a eu pour objectif (i) d'adapter les techniques de spectrométrie de masse déjà existantes pour l'analyse de la matière organique fossile (notamment par la sélection de la source d'ionisation atmosphérique la plus adaptée) mais également (ii) de développer un nouveau type de couplage entre la chromatographie d'exclusion stérique (SEC) et la spectrométrie de masse APPI-QTOF pour l'analyse des fractions peu polaires de hauts poids moléculaires. L'adaptation du l'APPI-QTOF a tout d'abord permis de mieux comprendre la réactivité de contaminants organiques polyaromatiques en présence de phases minérales. Le couplage SEC-APPI-QTOF a, quant à lui, permis d'améliorer les connaissances sur la structure des asphaltènes. Cependant, malgré la « simplification » rendue possible par la SEC, la très grande quantité d'informations reste difficile à interpréter et prend beaucoup de temps. Un modèle mathématique a donc été développé basé sur des analyses numériques et statistiques des spectres de masse, permettant de les comparer entre eux afin de distinguer l'origine des échantillons et de suivre l'impact de processus physico-chimiques (altérations naturelles - traitements de remédiation).

The development of analytical tools for organic geochemistry analysis has increased these past years. This development has allowed answering many questions about organic matter composition. However, many issues remain to be clarified including the characterization of high molecular weight fractions and monitoring the reactivity of organic matter. This thesis has focused on both (i) existing method improvements for fossil organic geochemistry analysis but also on (ii) developing a new type of coupling between the size exclusion chromatography (SEC) and the APPI-QTOF mass spectrometry for high molecular weight weakly polar fractions. Adjustments on APPI-QTOF mass spectrometry have allowed a better understanding of polyaromatic organic contaminant reactivity in presence of mineral matrices. The success of this coupling has allowed a better understanding of the structure of asphaltenes. However despite the "simplification" obtained by the SEC, the large amount of information remains difficult to interpret and time-consuming. A mathematical model has been developed based on numerical and statistical analysis of mass spectra, allowing direct comparison of mass spectra and being able to identify several types of information such as origins of samples, monitoring of physico-chemical processes and also the efficiency of soil recovery treatments as well as the identification of analytical protocols., Le développement des outils analytiques pour l'analyse de la matière organique complexe en géochimie organique a connu de nombreuses avancées ces dernières années. Ce développement a permis de répondre à un grand nombre de questions quant à la composition de la matière organique. Cependant, beaucoup des points restent encore à élucider comme notamment la caractérisation des fractions de hauts poids moléculaires ainsi que le suivi de la réactivité de la matière organique. Ce travail de thèse a eu pour objectif (i) d'adapter les techniques de spectrométrie de masse déjà existantes pour l'analyse de la matière organique fossile (notamment par la sélection de la source d'ionisation atmosphérique la plus adaptée) mais également (ii) de développer un nouveau type de couplage entre la chromatographie d'exclusion stérique (SEC) et la spectrométrie de masse APPI-QTOF pour l'analyse des fractions peu polaires de hauts poids moléculaires. L'adaptation du l'APPI-QTOF a tout d'abord permis de mieux comprendre la réactivité de contaminants organiques polyaromatiques en présence de phases minérales. Le couplage SEC-APPI-QTOF a, quant à lui, permis d'améliorer les connaissances sur la structure des asphaltènes. Cependant, malgré la « simplification » rendue possible par la SEC, la très grande quantité d'informations reste difficile à interpréter et prend beaucoup de temps. Un modèle mathématique a donc été développé basé sur des analyses numériques et statistiques des spectres de masse, permettant de les comparer entre eux afin de distinguer l'origine des échantillons et de suivre l'impact de processus physico-chimiques (altérations naturelles - traitements de remédiation).

Several cyanobacterial genera produce toxic secondary metabolites, the most well-known of which are the hepatotoxic microcystins (MCYSTs). Microcystin analyses in drinking water are a requirement of the Health Ministry (Regulation 518/2004) in Brazil, but this regulation does not establish which extraction and analytical method should be used; toxin quantification is usually carried out by ELISA (enzyme-linked immunosorbent assay) or HPLC (high performance liquid chromatography), the efficiency of which depends on the extraction method used. In this work a simple, fast and cheap method of extraction was developed for the isolation and identification of MCYSTs. For this, the strain Microcystis aeruginosa NPLJ-4, reported to be a MCYST-LR producer, was selected. Eight different treatments were tested to determine the best MCYST extraction. Samples were applied in LC-MS (liquid chromatography-mass spectrometry), ELISA and Q-TOF (quadrupole time-of-flight). The most efficient extraction was achieved by sonicating samples diluted in water. The proposed method permits rapid sample processing, and establishes an extraction method for both the analysis and identification of MCYST-LR and other variants. Vários gêneros de cianobactérias produzem metabólitos secundários tóxicos, entre eles as hepatotoxinas microcistinas. A análise de microcistinas em águas para abastecimento humano é uma exigência do Ministério da Saúde (Portaria 518/2004), mas essa portaria ainda não estabelece o método de extração e análise a serem usados e a quantificação da toxina é comumente realizada por ELISA ("enzyme-linked immunosorbent assay") ou HPLC (cromatografia líquida de alta eficiência), cuja eficiência depende do método de extração utilizado. Neste trabalho foi desenvolvido um método simples, rápido e barato de extração para o isolamento e identificação de microcistinas. Para isso, selecionou-se a linhagem Microcystis aeruginosa NPLJ-4 descrita como produtora de microcistina-LR. Oito diferentes tratamentos foram testados para determinar a melhor extração da toxina. As amostras foram analisadas por LC-MS (cromatografia líquida acoplada a espectrometria de massas), ELISA e Q-TOF ("quadrupole time-of-flight"). Os resultados mostraram que a melhor extração foi a que usou sonicação das amostras diluídas em água. O método proposto permite o processamento rápido das amostras e estabelece um método de extração para análise e identificação de microcistina-LR e outras variantes.

A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.

International audience; The final chemical structure of a newly synthesized protein is often only attained after further covalent modification. Ideally, a comprehensive proteome analysis includes this aspect, a task that is complicated by our incomplete knowledge of the range of possible modifications and often by the lack of suitable analysis methods. Here we present two recently discovered, unusual forms of protein glycosylation, i.e. C-mannosylation and O-fucosylation. Their analysis by a combined mass spectrometric approach is illustrated with peptides from the thrombospondin type 1 repeats (TSRs) of the recombinant axonal guidance protein F-spondin. Nano-electrospray ionization tandem-mass spectrometry of isolated peptides showed that eight of ten Trp residues in the TSRs of F-spondin are C-mannosylated. O-Fucosylation sites were determined by a recently established nano-electrospray ionization quadrupole time-of-flight tandem-mass spectrometry approach. Four of five TSRs carry the disaccharide Hex-dHex-O-Ser/Thr in close proximity to the C-mannosylation sites. In analogy to thrombospondin-1, we assume this to be Glc-Fuc-O-Ser/Thr. Our current knowledge of these glycosylations will be discussed.

Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1348-6) contains supplementary material, which is available to authorized users.