O fluconazol é um fármaco antifúngico utilizado na prevenção e tratamento de infecções micóticas. Atualmente, no mercado brasileiro, vários laboratórios farmacêuticos comercializam produtos a base de fluconazol na forma de cápsulas de 150 mg. Estes produtos são considerados similares e, portanto, teoricamente intercambiáveis, por conterem o mesmo princípio ativo nas mesmas dosagem e forma farmacêutica. No entanto, não existem estudos atestando a bioequivalência entre eles. Pretendeu-se, nesse trabalho, realizar avaliação biofarmacotécnica in vitro (cinética de dissolução) e in vivo (bioequivalência) de duas formulações do mercado nacional contendo fluconazol: Zoltec® 150 mg (laboratórios Pfizer Ltda.), considerado produto referência (R) e Flunazol® 150 mg (Laboratórios Sintofarma S.A.), considerado produto teste (T). Inicialmente, desenvolveu-se método para a análise da cinética de dissolução, já que não existe teste oficial de dissolução para formas farmacêuticas contendo fluconazol. Após padronização do método, avaliou-se a cinética de dissolução de cápsulas de fluconazol provenientes de dois lotes de R e dois lotes de T por meio dos parâmetros ks (constante de velocidade de dissolução) e t85% (tempo necessário para dissolução de 85% do fármaco presente na forma farmacêutica), derivados dos perfis de dissolução. Obteve-se ks de 0,1079 min-1 e 0,1377 min-1 para os dois lotes de R testados e 0,5421 min-1 para os dosi lotes de T, e t85% entre 15,09 min e 20,06 min para R e entre 5,64 min e 6,02 min para T. O ensaio de bioequivalência foi do tipo aleatório cruzado, com coleta de amostras de sangue até 96 horas após administração dos produtos R e T a 28 voluntários em jejum. Para quantificação do fluconazol em amostras de plasma desenvolveu-se e validou-se método simples, exato, preciso e sensível por cromatografia líqüida de alta eficiência (CLAE) sem utilização de padrão interno, e detecção em ultravioleta a 210 nm, após extração com solvente orgânico em meio básico. A bioequivalência entre os produtos foi determinada através da comparação dos parâmetros farmacocinéticos Cmax (concentração plasmática máxima), tmax (tempo necessário para Cmax) e AUCT (área sob a curva de decaimento plasmático) obtidos para R e T. Os resultados foram submetidos a análise estatística conforme recomendado pelo FDA-USA, determinando-se os intervalos de confiança 90% (I.C. 90%) para as relações entre Cmax de T e R e AUCT de T e R. Os valores médios de Cmax, tmax e AUCT para R e T foram, respectivamente: 3,64 mg/L e 3,75 mg/L; 2,96 h e 2,79 h; 153,33 mgh/L e 154,45 mgh/L. Os I.C. 90% para Cmax e AUCT foram, respectivamente, 101,06% a 105,45% e 97,96% a 103,36%. Concluiu-se que R e T são bioequivalentes, podendo ser administrados de forma intercambiável, sem prejuízo do efeito terapêutico. / Fluconazole is an antifungal agent widely used in the prevention and treatment of invasive fungal infections. Many brazilian pharmaceutical industries manufacture capsules containing 150 mg of fluconazole. As such products contain the same amount of the same therapeutically active ingredient in the same dosage form, they are considered to be interchangeable, indeed no bioequivalence study have been conducted to assess this. The present study was designed to perform in vitro (dissolution kinetics) and in vivo (bioequivalence) biopharmaceutical evaluation of two commercial products available in Brazil: Zoltec® 150 mg (Pfizer) as the reference product (R) and Flunazol® 150 mg (Sintofarma) as the test product (T). There is no official dissolution method for fluconazole dosage forms so initially a methos was developed and standardized for the evaluation of dissolution kinetics of fluconazole capsules. Dissolution kinetics for samples from two batches of R and two batches of T was analysed through ks (dissolution rate constant) and t85% (time for dissolution of 85% of the drug in the dosage form), obtained from dissolution profiles. Results showed ks values of 0,1079 min-1 and 0,1377 min-1 for the two tested batches of R and 0,5421 min-1 for both tested batches of T, and t85% values between 15,09 min and 20,06 min for R and between 5,64 min and 6,02 min for T. Bioequivalence assay was crossover and randomized. Blood samples were collected throughout a 96 hours period after administration of R and T to 28 fasting volunteers. A simple, accurate, precise and sensitive high-performance liquid chromatographic (HPLC) method without internal standard, and ultraviolet detection at 210 nm, was developed and validated for quantification of fluconazole im plasma samples after liquid-liquid extraction. Bioequivalence was assessed through pharmacokinetic parameters Cmax (peak plasma concentration), tmax (time to reach Cmax) and AUCT (área under the “plasma concentration vs time” curve) for R and T. Results were submitted to statistical analysis according to the FDA-USA and 90% confidence intervals (90% C.I.) were calculated for T and R Cmax ratios and T and R AUCT ratios. Average Cmax, tmax and AUCT values for R and T were, respectively: 3,64 mg/L and 3,75 mg/L; 2,96 h and 2,79 h; 153,33 mgh/L and 154,45 mgh/L. 90% C.I. for Cmax and AUCT were, respectively, 101,06% - 105,45% and 97,96% - 103,36%. Results show that R and T are bioequivalent and can be administered in an interchangeable way, without any prejudice of therapeutic effect.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-04T19:06:17Z No. of bitstreams: 1 Anderson da Silva Rosa.pdf: 3717734 bytes, checksum: 87724729c4a8c7bfe10174d0b64ea722 (MD5) / Made available in DSpace on 2016-04-04T19:06:17Z (GMT). No. of bitstreams: 1 Anderson da Silva Rosa.pdf: 3717734 bytes, checksum: 87724729c4a8c7bfe10174d0b64ea722 (MD5) Previous issue date: 2013-10-12 / Pesticidas são compostos sintéticos que possibilitam o aumento da produção e da qualidade dos produtos agrícolas através da diminuição das perdas geradas por organismos indesejados. No entanto, eles podem gerar um gradativo impacto ambiental devido a sua toxicidade. Resíduos de pesticidas e seus metabólitos têm sido frequentemente encontrados em águas subterrâneas e de superfície. Desta forma, a contaminação de corpos d'água pode afetar direta ou indiretamente a saúde humana e a integridade do ecossistema por induzir uma ameaça significativa em ambientes aquáticos e recursos de água potável. A descontaminação de águas contaminadas por pesticidas tem um custo elevado e envolve grande gasto de energia. A fitorremediação (o uso de plantas para descontaminação de compostos xenobióticos) ganhou popularidade como uma rentável, ambientalmente amigável e eficiente tecnologia insitu para uma variedade de poluentes e, entre eles, muitos pesticidas. O presente estudo avaliou o potencial de fitorremediação da alface (Lactuca sativa L.) em águas contendo uma mistura dos pesticidas quincloraque, 2,4-D, clomazone, propanil e tebuconazole na concentração de 50 μg/L através de cultivo hidropônico. Para tal fim, foi desenvolvido e validado um método multirresidual para a determinação destes pesticidas citados por cromatografia líquida de alta eficiência com detecção por arranjo de diodos (HPLCDAD) em água potável e de cultivo hidropônico. O método desenvolvido mostrou-se eficaz, confiável, com boa sensibilidade e repetibilidade para a determinação dos compostos de interesse. O método proporciona ainda, flexibilidade para incluir novos pesticidas e/ou seus produtos de degradação. Para a avaliação da fitorremediação, mudas de alface foram cultivadas e após dez dias os meios de cultivo foram fortificados com os pesticidas em estudo. Foram coletadas amostras da água de cultivo no dia da fortificação, no sétimo e décimo-quarto dia após o tratamento. Houve uma diminuição significativa das concentrações de quincloraque, tebuconazole, 2,4-D e clomazone, sendo os melhores resultados obtidos após sete dias para os dois primeiros compostos e quatorze dias para os últimos, respectivamente. Esse estudo mostra o potencial do cultivo hidropônico de alface para a fitorremediação de pesticidas em água. / Pesticides are synthetic compounds that enable an increased production and quality of agricultural products by reducing losses caused by unwanted organisms. However, they can generate a gradual environmental problem due to their toxicity. Pesticide residues and their metabolites have often been found in groundwater and surface. Thus, the contamination of water bodies may directly or indirectly affect human health and the ecosystem integrity by inducing a significant threat to aquatic environments and drinking water resources. The decontamination of water contaminated by pesticides is expensive and involves great expenditure of energy. The phytoremediation (the use of plants to decontaminate xenobiotics) has gained popularity as a low cost, environmentally friendly and efficient in situ technology for a variety of pollutants and, among them, many pesticides. The present study evaluated the potential for phytoremediation of lettuce to water containing a mixture of pesticides quinclorac, 2,4- D, clomazone, propanil and tebuconazole concentration of 50 μg/L through hydroponics. Therefore, a multirresidue method for determination of pesticides cited by high performance liquid chromatography, with diode array detection (HPLC-DAD), in drinking water and hydroponic medium was developed and validated. The method has proven effective, reliable with good sensitivity and reproducibility for determining the compounds of interest. The method provides further flexibility to add new pesticide and/or its degradation products. For the evaluation of phytoremediation lettuce seedlings were grown and after ten days, the culture media was fortified with the x pesticides under study. Water samples were collected from cultivation on fortification, on the 7th and 14th days after treatment. It was observed a significant decrease in concentrations of quinclorac, tebuconazole, clomazone and 2,4-D, and the best results were obtained after seven days for the first two compounds and for the past fourteen days, respectively. This study shows the potential of hydroponic cultivation of lettuce for phytoremediation of pesticides in water.

Oligonucleotides are synthetic strings of DNA or RNA used mostly for biochemical analysis and diagnostics. For them to be useful in these fields, a purity over 90% is most often required. However, when synthesizing these sequences, many “failures” (shorter sequences) are made in the step-wise process. The synthesized oligonucleotides need to therefore be purified. This is most often done with gel electrophoresis or liquid chromatography. These methods are, on the other hand, very time-consuming and laborious. Solid phase extraction (SPE) is a much faster purification method if optimized and it can be done with the standard cartridges as well as 96-well plates, that allow many samples to efficiently be run at the same time. With reversed phase (RP) SPE, the dimethoxytrityl (DMT) group, that is attached to the target at the final synthesis step, can be used for stronger retention to the bed sorbent and leaving only the target at the final eluting stage. The impurities without a DMT-on group, that do not adsorb to the sorbent, are washed away in earlier steps. The purpose of this study is to optimize an SPE method for purification of oligonucleotides. Two different cartridges, Clarity QSP (Phenomenex) and Glen-Pak (Glen Research) were used. The purity analysis and oligonucleotide identification were done using anion exchange - high performance liquid chromatography (AIE-HPLC) and time-of-flight mass spectrometry (TOF MS). To conclude, Clarity QSP achieved, at the most, a purity of 68.8% with the recommended SPE steps by Phenomenex. Alterations in the extraction procedure resulted in similar purity or lower. Glen-Pak reached a peak purity of 78.8% when doing a double salt wash of 5% ACN in 2 M sodium chloride and another double wash after detritylation with 1% acetonitrile. This method has to be further optimized in order to reach a purity of at least 90% to be useful in industrial settings.

Although the prevalence of substandard and counterfeit pharmaceutical products is a global problem, it is more critical in resource-constrained countries. The national medicines regulatory authorities (MNRA) in these countries have limited resources to cater for regular quality surveillance programmes aimed at ensuring that medicines in circulation are of acceptable quality. Among the reasons explained to hinder the implementation of these strategies is that compendial monographs are too complicated and require expensive infrastructures in terms of environment, equipment and consumables. In this study it was therefore aimed at developing simple, precise, and robust HPLC and HPTLC methods utilizing inexpensive, readily available chemicals (methanol and simple buffers) that can determine the APIs, other API than declared one, and which are capable of impurity profiling. As an outcome of this study, three isocratic and robust HPLC and two HPTLC methods for sulfadoxine, sulfalene, pyrimethamine, primaquine, artesunate, as well as amodiaquine have been developed and validated. All HPLC methods are operated using an isocratic elution mode which means they can be implemented even with a single pump HPLC system and standard C18 columns. The densitometric sulfadoxine/sulfalene and pyrimethamine method utilizes standard TLC plates as well as inexpensive, readily available and safe chemicals (toluene, methanol, and ethyl acetate), while that for artesunate and amodiaquine requires HPTLC plates as well as triethylamine and acetonitrile due to challenges associated with the analysis of amodiaquine and poorly the detectable artesunate. These HPTLC methods can be implemented as alternative to those requiring HPLC equipment e.g. in countries that already have acquired densitometer equipment. It is understood that HPTLC methods are less sensitive, precise and accurate when compared to HPLC methods, but this hindrance can easily be addressed by sending representative samples to third party quality control laboratories where the analytical results are verified using compendial HPLC methods on a regular basis. It is therefore anticipated that the implementation of these methods will not only address the problem of limited resources required for medicines quality control but also increase the number of monitored targeted antimalarial products as well as the number of resource- constrained countries participating in quality monitoring campaigns. Moreover, the experiences and skills acquired within this work will be applied to other API groups, e. g. antibiotics, afterwards. / Trotz der weltweiten Verbreitung gefälschter Arzneimittel und solcher, die nicht die deklarierte Menge an Wirkstoff enthalten, sind vor allem Entwicklungs- und Schwellenländer von dieser Problematik betroffen. Die Arzneimittelüberwachungs- bzw. Zulassungsbehörden dieser Länder verfügen nur über eingeschränkte Möglichkeiten, die Arzneimittelqualität regelmäßig zu überwachen und somit sicherzustellen, dass die im Markt befindlichen Medikamente eine gute Qualität aufweisen. Einer der Gründe hierfür ist unter anderem, dass die in Arzneibüchern beschriebenen Methoden oftmals sehr komplex sind und eine umfassende Laborausstattung, spezielle Geräte oder teure Chemikalien benötigen. In dieser Arbeit wurden einfache, genaue und robuste flüssigchromatographische Methoden entwickelt, die lediglich günstige, überall verfügbare Chemikalien (z. B. Methanol oder einfache Puffersalze) benötigen und mit denen der Gehalt des deklarierten Arzneistoffes, Arzneistoffverwechslungen sowie das Verunreinigungsprofil bestimmt werden kann. Es konnten drei isokratische, robuste flüssigchromatographische sowie zwei dünnschichtchromatographische Methoden zur Bestimmung von Sulfadoxin, Sulfalen, Pyrimethamin, Primaquin, Artesunat sowie Amodiaquin entwickelt und validiert werden. Alle flüssigchromatographischen Methoden arbeiten isokratisch, folglich können sie auch mit sehr einfachen HPLC-Geräten mit beispielsweise nur einem Pumpenkopf genutzt werden. Zudem werden nur einfache, kommerziell erhältliche C18-Säulen benötigt. Die densitometrischen Methoden für Sulfadoxin/Sulfalen sowie Pyrimethamin benötigen standardisierte Dünnschichtchromatographie-Platten sowie günstige, überall verfügbare und wenig toxische Chemikalien wie beispielsweise Toluol, Methanol oder Ethylacetat. Für die Methode zur Bestimmung von Artesunat und Amodiaquin werden Hochleistungsdünnschichtchromatographie-Platten und Triethylamin sowie Acetonitril benötigt. Dieser Umstand ist der Tatsache geschuldet, dass Amodiaquin und Artesunat sich anderweitig nur ungenügend trennen ließen. Die dünnschichtchromatographischen Protokolle können als Alternative zur HPLC eingesetzt werden, beispielsweise überall dort, wo bereits die entsprechenden Gerätschaften vorhanden sind. Natürlich weisen dünnschichtchromatographische Methoden im Vergleich zur Flüssigchromatographie eine geringere Sensitivität, Präzision und Richtigkeit auf, dies kann jedoch dadurch umgangen werden, die entsprechenden Methoden nur zum Screening zu verwenden und die zu analysierenden Proben anderweitig, z. B. in externen Laboratorien, detailliert zu untersuchen. Dort können beispielsweise Methoden aus gängigen Arzneibüchern verwendet werden. Durch die Implementierung der neu entwickelten Methoden kann zum einen das Problem schlecht verfügbarer Chemikalien umgangen werden und gleichzeitig die Anzahl an untersuchten Arzneimitteln erhöht werden. Dies ist ein wichtiger Beitrag zur Qualitätskontrolle in Ländern mit eingeschränkten Infrastrukturen.

<p>The objective for this master’s thesis was to test and evaluate two methods for determining the content of tannin in a pinebarkextract. The methods used at Södra for this previously have not been specific enough, only the amount of polyphenolics have been measured. One of these methods is a test based on the Stiasny test and it determines the extracts ability to form a gel with formaldehyde. When this test was carried out it showed that the extract did not contain much tannin. The other method used at Södra measures the amount of polyphenolic substances with a spectrophotometer. The result from this method showed that the content were 50 %.</p><p> </p><p>One of the two new methods that were evaluated determines the amount tannin present in the extract because of tannins ability to form a complex with proteins. According to this method, the amount tannin in the extract were 42,5 %, and 19,4 % of this in the form of tannic acid. These results are credible on basis of previous information about the extract.</p><p> </p><p>The other method uses a RP- HPLC where ellagic and gallic acid were used as standards. The amount ellagic acid in the extract was determined to 0,06 %, but the result from gallic acid was inconclusive. Further analysis is necessary to evaluate the obtained results and the methods reliability.</p><p> </p><p>If the metods is to be used on a regular basis, my recommendation would be to start evaluating the protein-binding method because this would be easier and less time-consuming than modifying the HPLC- method. The HPLC- method could on the other hand provide useful information about the extract, not only the amount tannin could be measured, but also which kind of tannin could be investigated.</p>

<p>The purpose of this study was to evaluate enantioselective analytical methods by separation of the enantiomers of four drugs (citalopram, zopiclone, tramadol and methylphenidate) and their metabolites. The analyses were performed with HPLC-UV with columns whose stationary phases were based on macrocyclic glycopeptides (Chirobiotic V, V2 and T) and polysaccharides (Lux Cellulose-1, Cellulose-2 and Amylose-2).</p><p>The Chirobiotic V column showed high selectivity for citalopram and its metabolites. High resolution was obtained using a mobile phase consisting of methanol, acetic acid and ammonia. High selectivity for the enantiomers of zopiclone and its metabolites were obtained on the Cellulose-2 column using a mobile phase consisting of acetonitrile and ammonium acetate buffer.</p><p>The enantiomers of tramadol were separated with the Amylose-2 column. However, changes in the pressure arose, probably caused by the additive NH₄HCO₃. When the analysis was repeated at a later occasion, reproducible results were not obtained. With the Cellulose-1 column, lower selectivity was obtained, resulting in unacceptably long analysis time.</p><p>Only a few analyses of methylphenidate were performed and the results indicated that the glycopeptide columns had higher selectivity for this compound than the polysaccharides.</p>

Antimony is naturally occurring in the environment. The assessment of human exposure to environmental antimony is limited. This research focuses on the determination of antimony in water, beverages, and fruit. First, we explored whether there is a correlation between arsenic and antimony in water samples with a wide range of arsenic and antimony concentrations. The results showed absent correlation. Second, we determined antimony concentrations in bottled beverages including bottled water, soft drinks, juices and alcoholic drinks from Canada. The results showed that the antimony in most of these samples were below the Health Canada Guideline (6 g/L) for drinking water except one alcoholic drink which contains 7 g/L antimony. Further analysis of lemons and oranges using high performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection demonstrated the presence of antimonycitrate species in these fruits, which has not been reported in literature.

Many mixtures important to research consist of hundreds or even thousands of individual components of interest. These types of mixtures are far too complex to separate by a single chromatographic dimension in any reasonable amount of time. However, if a multidimensional approach is used, where a complex mixture is separated by an initial dimension, simpler fractions of that separation are collected and each of those fractions are analyzed individually, highly complex mixtures can be resolved in relatively short amounts of time. This dissertation serves as a guide to multidimensional chromatography, in particular, two-dimension liquid chromatography. There are many aspects of multidimensional separations that have been investigated to show its aspects, drawbacks and potential ability to separate highly complex mixtures. Measurements for the performance of multidimensional chromatography, the effects of the first and subsequent dimensions and the approaches to pairing dimensions are shown with experimental examples. Fundamental and practical features of multidimensional chromatography are explained as well as theoretical discussions on current and future multidimensional chromatography performance. Experimentally, very high peak capacities were obtained (ca. 7000) and an algorithm to predict how to best optimize a two-dimensional separation based on the time used and performance was created for designing experiments.

Iodine is a biophilic element with one natural long-lived isotope, 129I (t1/2= 15.6 million years), and one stable isotope, 127I. The inventory of 129I in surface environments has been overwhelmed by anthropogenic releases over the past 50 years. The objective of this study is to utilize the elevated concentration and biophilic nature of 129I and the isotopic ratio of iodine (129I/127I) as a tracer of water mass movement and organic matter. Additionally, the significantly elevated values of 129I/127I could provide a geochronometer, similar to the way 14C is used, particularly for terrestrial organic matter that is less than 50 years old. A series of laboratory experiments and field investigations were carried out to characterize the dominant chemical forms of dissolved iodine, i.e., iodide (I-), iodate (IO 3-), and organic iodine (DOI) in natural waters. Sensitive methods were developed for the analysis of nanomolar quantities of 127I species in a variety of environmental systems using high performance liquid chromatography (HPLC) and an organic iodine decomposition technique, dehydrohalogenation. The potential use of 129I/127I as a hydrological tracer was evaluated through measurements of 129I and 127I, which were carried out in wells in the artificially recharged ground water basin of Orange County, California. Literature values of aquifer ages based on 3H/3He and δ18O tracer data, as well as time-series data of chloride and Santa Ana River flow rates over the past decade were compared to values for 129I and 127I. The iodine isotopes demonstrated a conservative behavior in these aquifers, suggesting that the observed variations of these isotopes reflect past river flow conditions during the time of recharge. The feasibility of using 129I/127I ratios to trace terrestrial organic matter across an estuary was tested. A novel analytical technique to determine 129I/127I ratios in DOI was developed for this investigation. The results of a Galveston Bay transect clearly show that 129I/127I ratios in DOI can remain elevated up to salinity of about 15, but that 129I/127I values of inorganic iodine species do not show any trend with change in salinity gradient due to fast isotopic and chemical equilibration in the estuarine waters.

The three thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat several diseases, including inflammatory bowel disease (IBD). They are pro-drugs and are believed to act through the formation of thioguanine nucleotides (TGNs). Other important metabolites are the methylthioinosine nucleotides (meTINs). These metabolites are active in the white blood cells (WBCs).Most patients respond well to the thiopurine drugs but up to a third have to modify or discontinue their treatment due to adverse events or a lack of therapeutic effects. This could be caused by inter-patient variability in the metabolism of the drugs. Therapeutic drug monitoring (TDM) of thiopurine nucleotides in red blood cells (RBCs) is used to guide treatment. Current routine assays measure the nucleotides after hydrolysation to nucleic bases and are therefore unable to distinguish between mono-, di-, and triphosphates. Recently it was shown that these assays failed to predict the clinical outcome in about 40% of the patients. It has been suggested that measuring thioguanosine triphosphate (TGTP) (believed to be the most active of the TGNs) separately might increase the clinical value.An assay suitable for measuring thioguanosine mono- (TGMP) and diphosphate (TGDP) and TGTP, as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) separately in RBCs in clinical samples has been developed. In clinical studies of 82 IBD patients, we found no correlation between the thiopurine dose and metabolite levels in RBCs, thus illustrating the importance of metabolite measurements in the TDM of thiopurines.The TGN peak measured by the routine assay during TDM of patients treated with thiopurines consisted of TGTP and TGDP with a small contribution from TGMP. The meTIN also consisted of mono-, di- and triphosphates, but in different proportions, indicating differences in the formation. The inter-individual differences in nucleotide distribution were very small and a strong correlation between the different nucleotides and their respective sums was observed. As a consequence, measuring the mono-, di- and triphosphates separately was not beneficial in predicting remission, which was confirmed by the results from the clinical study.Further research into the metabolism and mode of action of thiopurine drugs is needed to understand the inter-patient variability in response and metabolite formation. An assay suitable for such studies, measuring TGNs and meTINs in cultured cells, has also been developed.