A diferença na atividade terapêutica, farmacocinética e / ou farmacodinâmica entre os enantiômeros de fármacos quirais impulsionou a necessidade de estudar e desenvolver métodos para determinação exata e precisa da pureza enantiomérica dos produtos farmacêuticos. Inicialmente, os enantiômeros foram separados em escala analítica. As condições analíticas foram adaptadas para a escala semi-preparativa para a obtenção enantiômeros puros. Os enantiômeros do bisoprolol foram separados através de Cromatografia Líquida de Alta Eficiência. Foi adotado o sistema direto de separação, fase normal, utilizando coluna Chiralcel OD (250 x 4,6 mm id). A fase móvel foi composta por hexano: etanol: dietilamina (80:20:0.2, v / v / v), vazão de 1mL/min e detecção em UV a 273 nm. A separação dos enantiômeros (R)-bisoprolol e (S)-bisoprolol foi obtida com sucesso em escala analítica e semi-preparativa. Considerando as características de uma separação quiral, podemos concluir que os resultados são eficazes, por ser uma separação rápida e seletiva. / The difference in therapeutic activity, pharmacokinetics, and / or pharmacodynamics between enantiomers of chiral drugs has raised the need to study and develop methods for accurate and precise determination of enantiomeric purity of pharmaceutical products. Initially, the enantiomers were separation in analytical scale. The analytical conditions were scaled up to semi-preparative level to obtain pure enantiomers. The enantiomers of bisoprolol were separated with high-performance liquid chromatography. Direct separation system was adopted in normal phase mode using Chiralcel OD column (250 x 4.6 mm id). The mobile phase was composed of hexane:ethanol:diethylamine (80:20:0.2, v/v/v), flow rate of 1mL/min and UV detection was made at 273 nm. The separation of enantiomers, (R)-bisoprolol and (S)-bisoprolol was successfully obtained in analytical and semi-preparative scale. Considering the characteristics of a chiral separation, we can conclude that the results are effective, because it is a fast and selective separation.

A diferença na atividade terapêutica, farmacocinética e / ou farmacodinâmica entre os enantiômeros de fármacos quirais impulsionou a necessidade de estudar e desenvolver métodos para determinação exata e precisa da pureza enantiomérica dos produtos farmacêuticos. Inicialmente, os enantiômeros foram separados em escala analítica. As condições analíticas foram adaptadas para a escala semi-preparativa para a obtenção enantiômeros puros. Os enantiômeros do bisoprolol foram separados através de Cromatografia Líquida de Alta Eficiência. Foi adotado o sistema direto de separação, fase normal, utilizando coluna Chiralcel OD (250 x 4,6 mm id). A fase móvel foi composta por hexano: etanol: dietilamina (80:20:0.2, v / v / v), vazão de 1mL/min e detecção em UV a 273 nm. A separação dos enantiômeros (R)-bisoprolol e (S)-bisoprolol foi obtida com sucesso em escala analítica e semi-preparativa. Considerando as características de uma separação quiral, podemos concluir que os resultados são eficazes, por ser uma separação rápida e seletiva. / The difference in therapeutic activity, pharmacokinetics, and / or pharmacodynamics between enantiomers of chiral drugs has raised the need to study and develop methods for accurate and precise determination of enantiomeric purity of pharmaceutical products. Initially, the enantiomers were separation in analytical scale. The analytical conditions were scaled up to semi-preparative level to obtain pure enantiomers. The enantiomers of bisoprolol were separated with high-performance liquid chromatography. Direct separation system was adopted in normal phase mode using Chiralcel OD column (250 x 4.6 mm id). The mobile phase was composed of hexane:ethanol:diethylamine (80:20:0.2, v/v/v), flow rate of 1mL/min and UV detection was made at 273 nm. The separation of enantiomers, (R)-bisoprolol and (S)-bisoprolol was successfully obtained in analytical and semi-preparative scale. Considering the characteristics of a chiral separation, we can conclude that the results are effective, because it is a fast and selective separation.

The presented works aimed on the analysis of new impurities in APIs and medicinal products. Different subtypes of LC were coupled to suitable detection methods, i.e. UV and various MS techniques, depending on the chemical natures of the analytes and the analytical task. Unexpected impurities in medicinal products and APIs caused several scandals in the past, concomitant with fatalities or severe side effects in human and veterinary patients. The detection of nitrosamines in sartans led to the discovery of nitrosamines in various other drugs, of which the antibiotic rifampicin was analyzed in this work. An examination of the synthesis of rifampicin revealed a high potential for the formation of 4-methyl-1-nitrosopiperazine (MeNP). An LC-MS/HRMS method suitable for the quantification of MeNP was applied in the analysis of drugs collected from Brazil, Comoros, India, Nepal, and Tanzania, where a single dose of rifampicin is used in the post-exposure prophylaxis of leprosy. All batches were contaminated with MeNP, ranging from 0.7-5.1 ppm. However, application of rifampicin containing up to 5 ppm MeNP was recommended by the regulatory authorities for the post-exposure prophylaxis of leprosy. In the 1990s the aminoglycoside antibiotic gentamicin attracted attention after causing fatalities in the USA, but the causative agent was never identified unequivocally. The related substance sisomicin was recognized as a lead impurity by the Holzgrabe lab at the University of Würzburg: sisomicin was accompanied by a variety of other impurities and batches containing sisomicin had caused the fatalities. In 2016, anaphylactic reactions were reported after application of gentamicin. A contamination of the medicinal products with histamine, an impurity of the raw material fish peptone used upon the production, could be identified as the cause of the adverse effects. Batches of gentamicin sulfate, which had been stored at the University of Würzburg since the earlier investigations, were analyzed regarding their contamination with histamine to determine whether the biogenic amine was responsible for the 1990s fatalities as well. Furthermore, a correlation with the lead impurity sisomicin was checked. Histamine could be detected in all analyzed batches, but at a lower level than in the batches responsible for the anaphylactic reactions. Moreover, there is no correlation of histamine with the lead impurity sisomicin. Hence, the causative agent for the 1990s fatalities was not histamine and remains unknown. Another source of impurities is the reaction of APIs with excipients, e.g. the esterification of naproxen with PEG 600 in soft gel capsules. The influence of the formulation’s composition on this reaction was investigated by means of LC-UV. Therefore, the impurity naproxen-PEG-ester (NPEG) was synthesized and used for the development of a method suitable for the analysis of soft gel capsule formulations. Different formulations were stressed for 7 d at 60 °C and the relative amount of NPEG was determined. The formation of NPEG was influenced by the concentrations of water and lactic acid, the pH, and the drug load of the formulation, which can easily be explained by the chemistry behind esterification reactions. Keeping in mind the huge variety of sources of impurities, it might be impossible to predict all potential impurities of a drug substance/product. Targeted and untargeted approaches were combined in the impurity profiling of bisoprolol fumarate. Eight versions of an LC-HRMS method were developed to enable the detection of a maximum number of impurities: an acidic and a basic buffered LC was coupled to MS detection applying ESI and APCI, both in positive in negative mode. MS and MS/MS data were acquired simultaneously by information dependent acquisition. In the targeted approach, potential impurities were derived from a reaction matrix based on the synthesis route of the API, while the untargeted part was based on general unknown comparative screening to identify additional signals. 18 and 17 impurities were detected in the targeted and the untargeted approach, respectively. The molecular formulae were assessed based on the exact mass and the isotope pattern. Theoretical fragment spectra generated by in silico fragmentation were matched with experimental data to estimate the plausibility of proposed/elucidated structures. Moreover, the detected impurities were quantified with respect to an internal standard. / In den vorgestellten Projekten wurden neue Verunreinigungen in Arzneistoffen und Arzneimitteln untersucht. Verschiedene flüssigchromatographische Methoden wurden mit geeigneten Detektionsverfahren gekoppelt. UV-Detektion und verschiedene massenspektrometrische Techniken wurden in Abhängigkeit der chemischen Eigenschaften der Analyten und der analytischen Herausforderung ausgewählt. Eine Kontamination von Wirkstoffen bzw. humanen und veterinären Arzneimittel mit unerwarteten Verunreinigungen löste mehrere Skandale aus, die mit Todesfällen oder ernsthaften Nebenwirkungen einhergingen. Die Identifikation von Nitrosamin-Verunreinigungen in Sartanen führte zur Entdeckung von Nitrosaminen in verschiedenen anderen Wirkstoffen, z.B. im Antibiotikum Rifampicin, das in dieser Arbeit untersucht wurde. Eine Betrachtung der Rifampicin-Synthese offenbarte ein hohes Potenzial der Bildung von 4-Methyl-1-nitrosopiperazin (MeNP). Arzneimittel aus Brasilien, Indien, Nepal, Tansania und von den Komoren wurden mittels LC-MS/HRMS bezüglich ihres MeNP-Gehaltes analysiert. Alle untersuchten Chargen waren mit MeNP im Bereich von 0.7-5.1 ppm belastet. Rifampicin wird in den genannten Ländern unter anderem als Einzeldosis zur Postexpositionsprophylaxe von Lepra eingesetzt. Für diese Indikation empfehlen die Zulassungsbehörden die Verwendung von Rifampicin mit bis zu 5 ppm MeNP. Das Aminoglycosid-Antibiotikum Gentamicin löste in den 1990er Jahren Todesfälle in den USA aus. Die verantwortliche Verunreinigung wurde nie eindeutig aufgeklärt, doch die verwandte Substanz Sisomicin wurde durch den Arbeitskreis Holzgrabe an der Universität Würzburg als Leitverunreinigung identifiziert: Sisomicin ging mit einer Vielzahl von weiteren Verunreinigungen einher und Sisomicin-reiche Chargen hatten die Todesfälle ausgelöst. 2016 traten anaphylaktische Reaktionen nach Gentamicin-Anwendung auf. Histamin war als Verunreinigung von Fischpepton, einem Ausgangsmaterial der Produktion, in den Wirkstoff gelangt. Um zu überprüfen, ob Histamin auch für die Todesfälle in den 1990er Jahren verantwortlich war, wurden seit den früheren Untersuchungen gelagerte Gentamicin-Chargen bezüglich ihrer Verunreinigung mit Histamin untersucht. Außerdem wurde überprüft, ob es einen Zusammenhang mit dem Sisomicin-Gehalt gibt. In allen untersuchten Proben wurde Histamin gefunden, allerdings in einer geringeren Konzentration als in Chargen, die anaphylaktische Reaktionen ausgelöst hatten. Des Weiteren konnte kein Zusammenhang mit der Leitverunreinigung Sisomicin erkannt werden. Der Auslöser der Todesfälle in den 1990er Jahren war somit nicht Histamin, sondern bleibt weiterhin unbekannt. Ein weiterer Ursprung von Verunreinigungen ist die Reaktion des Wirkstoffs mit Hilfsstoff(en), z.B. die Veresterung von Naproxen mit PEG in Weichkapseln. Der Einfluss von Veränderungen der Formulierung auf diese Reaktion wurde mittels LC-UV untersucht. Die Verunreinigung Naproxen-PEG-Ester (NPEG) wurde synthetisiert und zur Entwicklung einer Methode zur Analyse von Weichkapsel-Formulierungen verwendet. Verschiedene Formulierungen wurden für 7 Tage bei 60 °C gestresst und deren relative Gehalte an NPEG bestimmt. Dabei war die Bildung von NPEG von der Wasser- und der Milchsäure-Konzentration, dem pH und dem Wirkstoffgehalt der Formulierung abhängig. Sämtliche Einflüsse konnten durch die Art der Reaktion (Veresterung) erklärt werden. Vor dem Hintergrund der vielfältigen Quellen für Verunreinigungen erscheint es unmöglich, alle potenziellen Verunreinigungen eines Wirkstoffs/Arzneimittels vorherzusagen. Gerichtete und ungerichtete Ansätze wurden daher für die Erstellung eines Verunreinigungsprofils von Bisoprololfumarat kombiniert. Um eine möglichst große Anzahl von möglichen Substanzen zu detektieren, wurden 8 LC-MS/HRMS-Methoden entwickelt: je eine saure und eine basische mobile Phase der LC wurde mit massenspektrometrischer Detektion mittels ESI und APCI, jeweils im positiv- und negativ-Modus kombiniert. MS- und MS/MS-Daten wurden simultan durch information dependent acquisition aufgenommen. Für den gerichteten Ansatz wurde eine Reaktionsmatrix auf Basis der Synthese des Wirkstoffs angefertigt und ausgehend davon potenzielle Verunreinigungen abgeleitet. Im ungerichteten Ansatz wurden mittels general unknown comparative screening zusätzliche Signale identifiziert. Der gerichtete Ansatz zeigte die Anwesenheit von 18, der ungerichtete von 17 Verunreinigungen. Summenformeln wurden anhand der exakten Masse und des Isotopenmusters der Signale bewertet. Die Plausibilität von Strukturformeln wurde mittels In-silico-Fragmentierung abgeschätzt, wobei experimentelle und theoretische Fragmentspektren in Einklang gebracht wurden. Außerdem wurden alle detektierten Verunreinigungen mittels eines externen Standards quantifiziert.

<p>UVOD: Učestalost kardiovaskularnih bolesti je visoka i predstavlja jedan od najče&scaron;ćih uzročnika morbiditeta i mortaliteta. Među evropskim zemljama najvi&scaron;i mortalitet od kardiovaskularnih bolesti beleži se u Srbiji. Hipertenzivna bolest je hronično oboljenje i često zahteva dugotrajnu medikamentoznu terapiju antihipertenzivnim lekovima. Osobe koje boluju od arterijske hipertenzije, koja je stabilna i dobro kontrolisana antihipertenzivnim lekovima, a čija doza nije menjana poslednje četiri nedelje, mogu biti davaoci krvi. MATERIJAL I METODE: Istraživanje je sprovedeno tokom 2012. godine na teritoriji grada Novog Sada i JužnoBačkog okruga. Prikupljeno je 450 uzoraka krvi od dobrovoljnih davalaca, oba pola, starosti od 18 do 65 godina, koji boluju od hipertenzivne bolesti, a koji u cilju terapije koriste antihipertenzivne lekove. Prikupljeni uzorci plazme analizirani su metodom tečne hromatografije visokih performansi (HPLC). REZULTATI: Analizom je utvrđeno prisustvo antihipertenzivnih lekova, iznad limita kvantifikacije, u 47 uzoraka. Prisustvo metoprolola je utvrđeno u 35, a bisoprolola u 12 uzoraka. U preostalim uzorcima zabeleženo je prisustvo ispitivanih lekova u količinama koje su bile iznad limita detekcije, ali ispod limita kvantifikacije. Detektovane koncentracije metoprolola se nalaze u terapijskom rasponu i za očekivati je da bi kod primaoca bili merljivi znaci efekta leka. Kod niskih detektovanih koncentracija bisoprolola verovatnoća pojave terapijskog efekta je mala i ne može se isključiti, jer bisprolol pokazuje terapijske efekte u rasponu koncentracija od 0,01 do 0,06 mcg/mL. ZAKLJUČAK: Vremenski period privremenog prestanka uzimanja antihipertenzivnih lekova za većinu ispitivanih lekova je odgovarajući i iznosi 24 časa pre davanja krvi. Period privremenog prekidanja terapije od 24 časa pre davanja krvi za metoprolol i bisoprolol potrebno je korigovati u smislu njegovog produženja i implementacija u Vodiče za selekciju i odabir davalaca krvi.</p> / <p>INTRODUCTION: The incidence of cardiovascular diseases is high and is one of the most common causes of morbidity and mortality among European countries. The highest mortality from cardiovascular diseases is recorded in Serbia. A hypertensive disease is a chronic condition and often requires a long-term medicamentous therapy using antihypertensive medications. Persons suffering from arterial hypertension, which is stable and well controlled when using antihypertensive medications, can be blood donors, providing that the dosage has not been changed in the last four weeks of the treatment. MATERIAL AND METHODS: The study was conducted during 2012. in the city of Novi Sad area and the South Backa District. 450 blood samples were collected from volunteer donors of both sexes, aged 18. to 65., all suffering from a hypertensive disease, who used antihypertensive medications for the purpose of the therapy. The collected plasma samples were analyzed using the method of high performance liquid chromatography (HPLC). RESULTS: The analysis revealed the presence of antihypertensive drugs, that were above the limit of quantification, in 47 samples. The presence of metoprolol was determined in 35 samples, and bisoprolol was present in 12 samples. The remaining samples recorded the presence of the tested drugs in amounts that were above the detection limit, but below the limit of quantification. The detected concentrations of metoprolol are in the therapeutic range, and it is to be expected that a recipient would show measurable signs of drug effect. Regarding the low detected values of bisoprolol, the probability of a therapeutic effect is small and can not be excluded, since bisoprolol shows therapeutic effects in the concentration range of 0.01 to 0.06 mcg/mL. CONCLUSION: The period of time to temporary withdraw the dosage of antihypertensive drugs in most investigated medications proved to be appropriate and is 24 hours prior to blood donation. The period of time to temporary interrupt therapy of 24 hours before giving blood for metoprolol and bisoprolol analysis should be corrected in terms of its extension and implementation in the Blood donors selection guide.</p>

The main aim of this study was to investigate the efficiency of wastewater treatment plant (WWTP) in České Budějovice for the elimination of selected pharmaceuticals (carbamazepine, diclofenac, atenolol, metoprolol, sotalol, bisoprolol, valsartan, verapamil and tramadol) over a long time period (March 2011 - February 2012). Time-proportional 24 hours pooled samples of wastewater from influent and effluent of the WWTP were used to assess the efficiency of WWTP. The concentrations of target compounds were determined by using in line SPE/LC-MS/MS analysis. The average annual concentrations in the effluent of WTP were in the range of 0,019 microgram/l (verapamil) to 1,00 microgram/l (atenolol). Average annual efficiencies of pharmaceutical elimination in WWTP based on pooled samples were found in the case of carbamazepine (-22 %), tramadol (-15 %), sotalol (-1 %), diclofenac (15 %), metoprolol (16 %), verapamil (43 %), bisoprolol (48 %) and valsartan (85 %). The statistical analysis of daily results in the winter and in the summer period showed significantly higher efficiency of the WWTP in the summer for 5 target compounds (diclofenac, atenolol, valsartan, sotalol and bisoprolol). Removal efficiency for the rest of pharmaceuticals did not show significant differences. Elevated temperature and longer irradiation period in summer can positively affect biodegradation or increased photolysis respectively.

This dissertation consists of six essays in health economics.The first essay, “Economic evaluations in health care: Basic principles and special topics”, serves as an introduction to economic evaluations in health care, including estimations of costs, health effects, and the discount rate. Special topics of interest for the rest of the studies are also discussed, e.g. the role of modelling in cost-effectiveness analysis, and methods for dealing with incomplete observations in clinical trial data. The main theme of the second essay, “Consumption and production by age in Sweden: Basic facts and health economic implications”, is a fairly detailed compilation of consumption and production figures by age in Sweden. The purpose of this is to use the difference between consumption and production in each age group as a measure of the average costs of added years of life in the general population. In economic evaluations of health care interventions, only future costs for related ill­nesses have typically been included in the analysis. However, the health economist David Meltzer has argued that future costs for un­related illnesses and general consumption should also be in­cluded in eco­nomic evaluations. Otherwise, the analysis will not be consistent with expected utility maximiza­tion. The third essay is entitled “The possibility of predicting health care costs in the future from predicted changes in age structure and age specific mortality: The case of Sweden”. Changes in the age structure, especially the growing number of elderly people, have raised concerns about increasing costs for health and elderly care in the future. However, the number of elderly per se is not the main problem, since the growing number of elderly people is a result of better health and hence lower morta­lity. The main purpose of the study is to investigate if future health care costs can be predicted based on forecasts of future changes in age structure and mortality rates. It is shown here that at least in Sweden and in the U.S., there is a linear relationship between age-specific mortality and age-specific health care costs. When these relationships are applied retrospectively to old data, however, the predictions are underestimates of the actual costs. These results are in line with earlier studies, which show that the future age structure is not likely to have a great impact on the future health care costs. The fourth essay is called “Cost effectiveness of bisoprolol in the treatment of chronic congestive heart failure in Sweden: Analysis using data from the Cardiac Insufficiency Bisoprolol Study II” (with Niklas Zethraeus and Bengt Jönsson). Treatment of heart failure with beta blockers was introduced in Sweden already in the 1970s, but it was not until the 1990s that large-scale clinical trials established the efficacy of beta blockers in reducing heart failure mortality. The study consists of an economic evaluation of the beta blocker bisoprolol added to standard treatment of chronic heart failure, compared with placebo added to the same standard treatment. The study raises a number of methodological issues. At the forefront are the inclusion of costs of added years of life, and the question of how to model health effects that extend beyond the clinical trial on which the economic evaluation is based. The results indi­cate that treatment with bisoprolol is cost-effective. A drawback of the analysis in the fourth study was that the expected survival after the end of follow-up was modelled deterministically. This makes it impossible to assess the uncertainty of the cost-effectiveness estimate in a realistic way. The fifth essay is entitled “Assessing uncertainty in cost-effectiveness analysis by combining resampling of clinical trial data with stochastic modelling: The economic evaluation of bisoprolol for heart failure revisited”. Here, the drawback with the fourth study that was mentioned above is addressed by using resamp­ling of the clinical trial data in combination with stochastic modelling of the expected survival after the end of follow-up in the clinical trial. The methodology is inspired by the bootstrap method, which is a simulation technique whereby various statistics, like the mean and variance, can be estimated through repeated resampling from the original sample. The difference from the traditional boot­strap method is that resampling of observations from the clinical trial data is combined with stochastic modelling of the expected remaining lifetime of the patients who were alive at the end of the clinical trial. Cost-effectiveness acceptability curves for treatment of heart failure with bisoprolol were obtained as a result of the analysis. The sixth essay, “Survival analysis techniques for estimating the costs attributable to head and neck cancer in Sweden”, concerns the estimation of average treatment cost attri­butable to a disease when the data contain censored, i.e. incomplete, observations. For various reasons, censored observations are common in medical and epidemiological studies. As a result, the length of the survival time or the size of the costs for those who are alive at the end of follow-up are not exactly known. This is of course problematic if we want to estimate the average survival time or the average cost for all patients, both survivors and non-survivors included. In this study, the Kaplan-Meier sample-average estimator is used for overcoming the problem with censored observations. It is a method that has been proposed specifically for handling censored cost data. / Diss. Stockholm: Handelshögsk., 2002

Diss. Stockholm: Handelshögsk., 2002.