M. Tech. Chemistry. / Aims to evaluate, compare and modify existing methods and investigate new methods for the qualitative and quantitative determination of melamine. Specific aims were to: evaluate and modify an existing GC-MS15 and HPLC-DAD method16, published by US FDA, for the determination of melamine in pet food and baby milk powder formulae ; develop a novel HPLC fluorescence detection (FLD) method for the determination of melamine in pet food and baby milk powder formulae ; compare these methods to a commercial ELISA detection method for the determination of melamine in pet food samples previously implicated in the poisoning of dogs ; apply NIR spectroscopy with subsequent multivariate analysis to rapidly determine melamine in pet food; and compare the different methods statistically with respect to their analytical performances.

published_or_final_version / Chemistry / Master / Master of Philosophy

In natural dendroflora of Lithuania willow genus (Salix L.) is abundant in species amount. Its bark contains active materials which are known as possessing anti-inflammatory properties since former times. Bark extracts are contained in analgesic as well as antirheumatic preparations. Its therapeutic effectiveness is associated with salicine which turns into salicylic acid. Willow bark is a know phytotherapeutic precursor of aspirin. To use more natural and not synthetic materials in the 21st century the methods for extraction of active materials for medicine are being sought. Thus, the studies on plant groups possessing the most intense anti-inflammatory properties became urgent. The amount of salicin varies among different species. Acording to the European Pharmacopoea, minimal amount of 1,5% of salicin is required in Willow bark. The aim of the study was to investigate salicylates amount variation in willow bark of different Salix species growing in Lithuania. The studies were carried out using the routine methods of pharmacopeia. After extraction analysis was performed by high performance liquid chromatography (HPLC) method. The first step of study was made among 3 samples of each plant, colected at different growing period (1-st year autumn, 2-nd year autumn and 2-nd year spring). Comparison of the quantities of the salicylic derivatives depending upon age of the plant and time of collection shows, that plants at two years of growth and collected in autumn accumulate... [to full text] / Natūralioje Lietuvos floroje gluosnių (Salix L.) rūšys yra plačiai paplitusios. Gluosnio žievė – nuo seno žinoma kaip priešuždegiminiu poveikiu pasižyminti vaistinė augalinė žaliava. Jos ekstraktai įeina į priešreumatinių ir analgezinių preparatų sudėtį. Terapinis veikimas yra susijęs su salicinu, kuris organizme virsta salicilo rūgštimi. Gluosnio žievė yra fitoterapinis aspirino pirmtakas. XXI amžiuje vis didesnis dėmesys krypsta į natūralius, o ne sintetinius preparatus. Tokiu būdu, augalų, pasižyminčių terapinėmis savybėmis tyrimai įgyja didelę reikšmę. Skirtingų rūšių gluosnių žievėje, kaupiamas salicino kiekis skiriasi. Europos farmakopėjoje reglamentuojamas minimalus salicino kiekis yra 1,5%. Šio tyrimo tikslas buvo Ištirti Lietuvoje augančių gluosnio rūšių salicilatų kiekio įvairavimą vaistinėje augalinėje žaliavoje (žievėje). Darbo uždaviniai- atlikti įvairių Lietuvoje augančių gluosnio rūšių salicilatų kiekio analizę, palyginti salicilatų kiekį gluosnio žievėje, atsižvelgiant į augalo amžių bei rinkimo laiką bei įvertinti gautus rezultatus, numatomą terapinį veikimą. Tyrimai atlikti naudojant farmakopėjinius analizės metodus. Žievės ekstraktai buvo tiriami efektyviosios skysčių chromatografijos metodu.Šio tyrimo pirmo etapo metu išanalizuota 12 gluosnių taksonų. Buvo ištirti vienerių ir dviejų metų, taip pat pavasarį ir rudenį rinkti žievės pavyzdžiai. Didesni kiekiai aktyvių medžiagų rasta rudenį ir 2 metų amžiaus gluosnių žievės pavyzdžiuose. Antrajame tyrimo... [toliau žr. visą tekstą]

Using a model system of glycosylated and aglycon standards consisting of rutin and quercetin respectively, and a series of pre-packed solid phase extraction cartridges, including C18 Extra-Clean, DSC-18, DPA-6S, Oasis HLB and Amberlite XAD-2. The experimental findings also showed that use of a commercial hesperinidase preparation, resulted in adequate hydrolysis of the glycosylated standard rutin. Based on these findings, the phenolic compounds and flavonoids from maple sap and syrup were separated using the Amberlite XAD-2 column, where the glycosylated fractions eluted with 60% aqueous methanol solution and the aglycon fractions eluted with a methanol:acetonitrile mixture (1:1, v/v). The recovered glycosylated fractions were subjected to enzymatic hydrolysis using the hesperinidase preparation and the liberated phenolic compounds and flavonoids, as well as the sugar components were analyzed by high-performance liquid chromatography (HPLC).

Comparative high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GC) analyses of selected phenolic and flavonoid standards were developed using a wide range of detectors, including ultraviolet diode-array (UV-DAD) and electrochemical (EC) detectors for HPLC and flame ionization detector (FID) and mass spectrometry (MS) for GC. The results demonstrated that the limits of detection obtained with HPLC-EC analysis were 10 to 500-times higher for phenolic acid standards and 2 to 50-times higher for flavonoid standards than those obtained with the HPLC-UV analysis. HPLC-EC was more sensitive than GC/FID for all investigated standards, especially for vanillin and syringaldehyde. The results indicated that GC/FID/MS analysis of phenolic and flavonoid standards was more efficient than that of HPLC, providing a fast analysis with better resolution and baseline separation of all standards with minimum co-elution. The only co-elution encountered in GC/FID was with coniferol and p-coumaric acids. For HPLC analysis, (-)-epicatechin, caffeic and homovanillic acids were co-eluted at 28.04 min and sinapic and ferulic acids at 34.57 min. Phenolic compounds and flavonoids were extracted from maple sap and maple syrup with ethyl acetate and the recovered compounds were subjected to HPLC and GC analyses. Tentative identification of phenolic compounds and flavonoids in maple sap and maple syrup indicated the presence of protocatechuic acid, hydroxycinnamic acid derivatives, (+)-catechin, (-)-epicatechin, vanillin, coniferol, syringaldehyde, flavanols and dihydroflavonols related compounds. In addition, the identification by GC/MS of protocatechuic acid, vanillin, syringaldehyde, coniferol and p-coumaric acid was made by comparing mass spectrum characteristics of individual peak from total ion chromatogram (TIC) to that of standard compounds. The seasonal variation of selected phenolic compounds and flavonoids present in maple sap and maple syrup was also invest

Three direct interfaces for coupling high performance liquid chromatography (HPLC) with atomic absorption spectrometry (AAS) were developed and optimized for the determination of ionic organolead, organoselenium and organoarsenic compounds. The first all-quartz interface consisted of a thermospray nebulizer and a flame microatomizer in which ionic alkyllead analytes (R$ sb{ rm n}$Pb$ sp{ rm (4-n)+};$ R = CH$ sb3,$ C$ sb2$H$ sb5)$ were atomized by a methanol (from HPLC eluent)-oxygen kinetic flame, and channeled in a quartz tube (atom keeper) mounted into the AAS optical beam. Alternately, the classical electrothermal atomization technique for organolead species (quartz furnace under hydrogen atmosphere) was coupled with a post-column derivatization-volatilization apparatus based on the ethylation of ionic alkylleads by sodium tetraethylborate. The limits of detection provided by these two approaches were 1.0-3.4 ng and 0.10-0.15 ng, respectively. Arsonium ((CH$ sb3) sb3$RAs$ sp+;$ R = CH$ sb3,$ CH$ sb2$CH$ sb2$OH, CH$ sb2$COOH) and selenonium ((CH$ sb3) sb2$RSe$ sp+;$ R = CH$ sb3,$ CH$ sb2$CH$ sb2$OH) species were quantified using a novel HPLC-AAS approach based on a direct coupling of three processes: thermospray nebulization, thermochemical hydride generation using hydrogen gas, and diffuse flame atomization. Direct evidences for the thermochemical hydride generation process was obtained by injecting (CH$ sb3) sb3$SeI and SeO$ sb2$ into the interface and capturing the gaseous end products in liquid chemical traps specific for SeH$ sb2$ and Se(IV). Both analytes were derivatized to SeH$ sb2$ only in the presence of hydrogen in the interface. Reverse- and normal-phase high pressure liquid chromatographic methods were also developed and adapted for the HPLC-AAS analyses of alkyllead, arsonium and selenonium compounds in real samples. The limit of detection of the arsonium and selenonium cations were 7.6-13.3 ng and 31.0-43.9 ng, respectively.

A direct injection nebulizer (DIN) was constructed in our laboratory and was evaluated as an interface between a liquid chromatography column and an inductively coupled plasma-atomic emission spectrometer (ICP-AES). Optimum operating conditions, detection limits and reproducibility of the DIN closely matched literature data for a somewhat different commercial device. In addition, when using the DIN for sample introduction, the ICP detection exhibited uniform response towards phosphorous compounds of different volatilities. / Department of Chemistry

Die DNA-Sequenzierung nimmt in der molekulargenetischen Diagnostik seit vielen Jahren einen großen Stellenwert ein. Zeit- und kosteneffektivere Methoden wie die DHPLC wurden seither für verschiedene Gene etabliert. Ziel dieser Arbeit war die Etablierung der DHPLC für das ATRX-Gen bei Patienten mit Verdacht auf das ATRX-Syndrom. Nach erfolgreicher Etablierung der DHPLC sollten im Rahmen dieser Arbeit 38 Patientenproben des Instituts für Humangenetik der Universität Leipzig mit Verdacht auf das ATRX-Syndrom mittels DHPLC und Sequenzierung untersucht werden. Die Etablierung der DHPLC gelang in der vorliegenden Arbeit für alle - das ATRX-Gen vollständig umspannenden - 42 Fragmente. Jede der vorliegenden Sequenzvariationen konnte nach Abschluss der Arbeit detektiert werden. Unter Verwendung von Maxima® stellten sich initial 22 von 24 verschiedenen Sequenzvariationen zum Wildtyp different dar. Die verbleibenden zwei Mutationen p.R246C und p.A238P im Fragment 9 wurden unter Verwendung höherer Temperaturen, eines kürzeren Fragmentes oder anderer Polymeraseenzyme (AmpliTaq Gold® bzw. HighFidelity) detektiert. Nach Abschluss der Etablierung der DHPLC konnte eine Sensitivität und Spezifität von 100% erreicht werden. In den Patientenuntersuchungen des Instituts für Humangenetik der Universität Leipzig fanden sich bei 38 Patientenproben vier verschiedene als benigne beschriebene Polymorphismen bei insgesamt 19 Patienten, ein noch nicht veröffentlichter Polymorphismus im Intron 26 sowie eine bis dato nicht beschriebene und als pathogen einzustufende Mutation im Exon 34. In 100 Kontrollen der DNA-Bank des Instituts für Humangenetik der Universität Leipzig konnte der bisher nicht publizierte Polymorphismus im Intron 26 sieben Mal gefunden werden. Die bis dato nicht beschriebene Deletion p.2385_2395del im Exon 34 führt zu einem vorzeitigen Abbruch des ATRX-Proteins und ist somit als sicher pathogen einzustufen. Die Untersuchung der Mutter des Patienten konnte den Konduktorenstatus nachweisen. Für die Familie des betroffenen Patienten konnte mit der vorliegenden Arbeit die Diagnose des ATRX-Syndroms gesichert werden. Die DNA-Proben der Patienten bei denen keine Mutation nachgewiesen werden konnte, sollten bei weiterhin dringendem Verdacht auf das ATRX-Syndrom mittels qRT-PCR bzw. MLPA untersucht werden, um große Deletionen, Insertionen oder Duplikationen auszuschließen. Mithilfe dieser Arbeit gelang die Etablierung der DHPLC für das ATRX-Gen als ökonomische, sehr sensitive und effiziente Methode zur Diagnostik des ATRX-Syndroms. Die Entscheidung, in welcher Form und mit welcher Methode DNA-Proben bei Verdacht auf ATRX-Syndrom untersucht werden, bleibt jedoch eine individuelle Entscheidung jedes Instituts unter Betrachtung der jeweiligen Gegebenheiten vor Ort.

Rykštenė yra daugiametis augalas, priklausantis astrinių šeimai. Ji pasižymi keliais farmakologiniais veikimais, tokiais kaip skatinančiu diurezę, priešuždegiminiu, analgetiniu, antispazmolitiniu, antibakteriniu ir antigrybeliniu, taip pat antioksidaciniu ir priešvėžiniu aktyvumu. Iš rykštenės žaliavos gaminami vaistai skatinantys diurezę. Ji taip pat įeina į Lietuvoje gaminamos arbatos „Diuretiko“ (UAB „Acorus Calamus”) sudėtį. Šio darbo tikslas - atlikti polifenolinių rūgščių kokybinę ir kiekybinę analizę rykštenės ekstraktuose taikant efektyviąją skysčių chromatografiją. Buvo paruošti Solidago virgaurea ir Solidago gigantea lapų ir žiedų ekstraktai naudojant vandeninį metanolio tirpalą (70%). Jie buvo analizuojami ESC metodu. Detekcijai naudojamas fotodiodų matricos detektorius. Gauti rezultatai buvo lyginami su standartinių junginių (chlorogeno ir neochlorogeno rūgščių) duomenimis. Polifenolinės rūgštys nustatomos bangos ilgiui esant 324nm. Buvo identifikuotos dvi polifenolinės rūgštys: chlorogeno ir neochlorogeno. Solidago virgaurea chlorageno rūgšties yra 1.31%, o neochlorogeno rūgšties – 0.06%. Solidago gigantea chlorogeno rūgšties procentinis kiekis yra 2.35%, o neochlorageno rūgšties – 0.02%. / Goldenrod is a perennial plant belonging to the Asteraceae family. It has several pharmacological functions, such as promoting diuresis, anti-inflammatory, analgesic, antispazmolitiniu, antibacterial and antifungal as well as anti-oxidative and anti-tumor activity. Of goldenrod made a medicaments stimulating diuresis. It also includes in production of tea „Diuretiko“ (UAB „Acorus Calamus”) composition in Lithuanian. The aim of this research was to make polyphenolic acids qualitative and quantitative analysis of the goldenrod extracts using high-performance liquid chromatography. Was prepared Solidago virgaurea and Solidago gigantea leaves and flowers extracts of aqueous methanol (70%). They were analyzed in HPLC method. For detection was used photodiode array detector. The results were compared with standard compounds (chlorogenic and neochlorogenic acids) data. Polyphenolic acids determined at the wave length of 324nm. Has been identified two polyphenolic acids: chlorogenic and neochlorogenic. In Solidago virgaurea chlorogenic acid is 1.31% and neochlorogenic acid - 0.06%. In Solidago gigantea chlorogenic acid percentage is 2.35%, and neochlorogenic acid - 0.02%.

Nuolatos plečiantis maisto papildų rinkai ir augant naujai sukuriamų papildų kiekiui būtina sukurti greitas, tikslias ir efektyvias papildų analizės metodikas, norint užtikrinti į rinką tiekiamų farmacinių prepartų kokybę. Vitamino E nustatymui efektyviosios skysčių chromatografijos būdu yra sukurta nemažai įvairių metodikų, tačiau svarbu žinoti, kuri metodika yra efektyviausia būtent vitamino E preparatams. Tyrimo tikslas - atlikti kokybinį ir kiekybinį tokoferolio acetato nustatymą pritaikant efektyviosios skysčių chromatografijos metodą bei surasti efektyviausią tokoferolio acetato ekstrakcijos metodiką vaistinių preparatų analizei. Tyrimo uždaviniai - surinkti ir apibendrinti informaciją apie vitaminą E, jo poveikį organizmui, maisto papildus su tokoferolio acetatu bei jų analizę; optimizuoti metodiką tokoferolio acetato preparatų analizei efektyviosios skysčių chromatografijos būdu ir ją validuoti; atlikti tokoferolio acetato preparatų (maisto papildo ir vaistinio preparato) paruošimą analizei įvairiomis ekstrakcijos metodikomis; išanalizuoti gautus duomenis ir padaryti išvadas apie efektyviausias ekstrakcijos metodikas bandinių paruošimui bei skirtumus tarp vaistinio preparato ir maisto papildo analizės. Metodas – tyrimas atliktas chromatografine sistema su fotodiodų matricos detektoriumi. Mobilioji fazė – metanolis:distiliuotas vanduo (95:5), tekėjimo greitis – 1.2 ml/min, bandinio tūris – 10 µl, trukmė – 14 min, bangos ilgis – 282 nm. Išbandytos preparatų... [toliau žr. visą tekstą] / The food supplements market is constantly growing and new products are entering it everyday. In order to assure the quality of these pharmaceuticals it is important to develop methods of analysis which are fast, precise and effective. There are many methods created for the analysis of vitamin E using high-performance liquid chromatography so it is important to know which of them are the most effective for vitamin E food supplements. The aim – to perform a qualitative and a quantitive assey of tochopherol acetate using high performance liquid chromatography and to find the most effective method of extracting tocopherol acetate from food suplements. The tasks – to collect and to summarize the data on vitamin E, its effects on human body, on the supplements containing tocopherol acetate and their analysis; to develop a high-performance liquid chromatography method of analysis suitable for the pharmaceuticals containing tocopherol acetate ant to validate it; to perform a tocopherol acetate (a food supplement and a drug) samples preparation using different techniques chosen; to analyze the data collected and to make conclusions about the most effective way of preparing samples for the analysis and about the differences between a drug and a food supplement analysis. The method – the study was conducted by high-performance liquid chromatography using a photodiode matrix detector. Mobile phase – methanol:purified water (95:5), flow-rate – 1.2 ml/min, injection volume – 10 µl... [to full text]