Das Ziel vorliegender Arbeit war die Entwicklung einer HPLC-MS(/MS)-Methode, die im Rahmen der pharmazeutischen Qualitätskontrolle zur direkten Quantifizierung von Aminosäuren (AS) in Infusionslösungen angewendet werden kann. Die Zielset-zung schloss eine Validierung innerhalb der für die Zweckbestimmung vorgesehenen Grenzen ein. Im Rahmen der Methodenentwicklung wurde das ESI-MS/MS-Fragmentierungs-muster von 21 Aminosäuren, von 20 stabil-isotopenmarkierten Aminosäuren, die als interne Standards verwendet wurden, sowie von einigen weiteren Substanzen bestimmt. Nach Kenntnis von Precursor- und Produktionen erstellte man eine SRM-Methode zur spezifischen MS/MS-Analyse. Dabei wurden durch das jeweilige Frag-mentierungsmuster bedingte Interferenzen bei den zu untersuchenden Aminosäuren bestimmt, die bei der zu erarbeitenden HPLC-MS-Methode beachtet werden mussten. Die Methodenentwicklung zur HPLC-MS-Analytik von underivatisierten AS umfasste mit der RP-HPLC unter Verwendung eines Ionenpaarreagenzes (IP) und der hydrophilen Interaktionschromatographie (HILIC) zwei verschiedene chromatographi-sche Ansätze. Bei der Anwendung der RP-HPLC ergaben sich Probleme. Die Verwendung eines IP, im vorliegenden Fall TDFHA (Tridecafluorheptansäure), führte zu langen Equilibrierungs-, Re-Equilibrierungs- und Spülzeiten und damit bei zwar relativ kurzer HPLC-Laufzeit zu einem aber insgesamt hohen Zeitaufwand. Gleich-zeitig war die LC-MS-Anlage auf diese Anwendung fixiert, da das Ionenpaarreagenz das Gerät stark verschmutzte und dadurch andere Analysen erheblich störte. Zudem waren die Retentionszeiten der Analyten trotz langer Equilibrierungszeiten schlecht reproduzierbar, so dass eine solche Methode im Rahmen der pharmazeutischen Qualitätskontrolle schwer validierbar wäre. Weiterführende Untersuchungen erfolgten daher nicht. In nachfolgenden Studien mit der HILIC wurden verschiedene Einflussparameter (Anteil organischer Phase im Fließmittel, pH-Wert des Fließmittels, Temperatur der Säule, Pufferkonzentration im Fließmittel, Gradientenelution) auf die Trennung der AS an einer ZIC®-HILIC-Säule untersucht. Durch Optimierung der Parameter wurde so eine HILIC-HPLC-Methode entwickelt, bei der 21 AS und 20 ihrer isotopen-markierten Referenz-AS innerhalb von 20 min eluierten. Diejenigen AS, bei denen im Rahmen der Fragmentierungsstudien Interferenzen aufgrund gleicher bzw. ähnlicher Massen der Precursor- bzw. Produktionen aufgetreten waren, wurden chroma-tographisch getrennt. Gleichzeitig hat sich die SIM-Analyse als anwendbar erwiesen. Die Anwendung des spezifischeren SRM-Modus und damit der Tandem-Massenspektrometrie war nicht erforderlich. Im Rahmen der nachfolgenden Studien zur Validierung ergab sich, dass die entwickelte Methode über einen weiten Bereich eine lineare Abhängigkeit zwischen Konzentrations- und Messwerten zeigte. Für drei der 21 AS (NAcCys, NAcTyr, Pro) wurde die quadratische Regression mit dem Anpassungstest nach Mandel als geeig-neteres Regressionsmodell ermittelt. Bei Untersuchungen zur Wiederfindung wurde ein Einfluss der Matrix-Lösung der Infusionslösung festgestellt, der zu Abweichungen hinsichtlich des Quotienten AreaAS / AreaIS führte, so dass eine Quantifizierung innerhalb der geforderten Grenzen bei Kalibrierung über reine Standardlösungen nicht möglich war. Die Validierung wurde daher nachfolgend in der Matrixlösung durchgeführt. Dabei wurde gezeigt, dass mit der entwickelten HILIC-HPLC-MS-Methode Aminosäuren in Infusionslösungen mit hoher Präzision und Richtigkeit bestimmt werden können. Neun der 21 untersuchten AS konnten im Bereich von 30% - 350%, zehn weitere im Bereich von 50% - 350% innerhalb der zur Gehaltsbestimmung von pharmazeutischen Formulierungen vorgeschriebenen Grenzen (Wiederfindung Einzelbestimmung: 98% -102.0%, Mittelwert einer Dreifachbestimmung: 98.5% – 101.5%) quantifiziert werden. Für His und Phe gelang allerdings keine Quantifizierung innerhalb der Akzeptanzkriterien, wobei der Grund hierfür in weiteren Studien geklärt werden müsste. Mit der entwickelten Methode ist damit eine gleichzeitige Quantifizierung verschiedener AS-Infusionslösungsformulierungen möglich, die sich bei gleicher Matrix in der Konzentration an AS unterscheiden. Beispielsweise seien hier die Formulierungen „Aminoplasmal® E 5% / 10% /15%“ genannt, die mit der validierten Methode erfassbar sind. Die Probenvorbereitung beschränkt sich dabei auf den Zusatz der IS-Formulierung zur Infusionslösung und einen Verdünnungsschritt. Die Quanti-fizierung erfolgt über eine 5-Punkt-Kalibriergerade, die aus einer AS- und IS-Standardmischung, nach Zusatz der einfach herzustellenden Elektrolyt-Matrix, erstellt wird. Die Analysenzeit der HPLC-MS-Methode beträgt einschließlich Equilibrie-rungszeit 35 min und ist damit deutlich kürzer als die 120 min, die bei der nach wie vor zur AS-Analytik allgemein gebräuchlichen Ionenaustauschchromatographie mit Ninhydrin-Nachsäulenderivatisierung anzusetzen sind. / The aim of this study was to develop an HPLC-MS(/MS) method to be used within pharmaceutical quality control for the direct quantitation of amino acids (AS) in infusion solutions. The validation within the limits of the intended purpose was part of the objective. In the course of the method development the ESI-MS/MS fragmentation pattern of 21 amino acids, 20 stable isotope labelled amino acids, which were used as internal standards, and some further substances were determined. By knowing precursor- and product ions a SRM-method for specific MS/MS analysis was created. Interferences due to the respective fragmentation pattern within the analytes were detected, which had to be considered during the following HPLC-MS method development. The HPLC-MS method development for the analysis of underivatized AS comprised two different approaches: the RP-HPLC using an ion-pair reagent (IP) und the hydrophilic interaction chromatography (HILIC). Problems occurred using the RP-HPLC. Due to the IP, in this study TDFHA (tridecafluoroheptanoic acid), long equilibration-, re-equilibration and rinsing-times were necessary so that this method is very time consuming despite short HPLC run times. Because of the contamination with TDFHA the use of the LC-MS system was limited. Furthermore, despite long equilibration times, retention times of the analytes were poorly reproducible. A validation of the method within the requirements of the pharmaceutical quality control would be therefore hardly to perform. Thus, further studies were not carried out. The following studies were focused on HILIC. Different parameters (percentage of organic solvent in the eluent, pH of the eluent, column temperature, buffer concentration in the eluent, gradient elution) were checked concerning their effects on the separation of AS on a ZIC®-HILIC column. By optimizing the parameters, a HILIC-HPLC method was developed, by which 21 AS and 20 of their stable labelled isotopes were eluted within 20 min. All AS, which interfered with other AS due to their fragmentation pattern, were chromatographically separated. Moreover, the SIM mode was found to be suitable for the separation of AS, thus avoiding SRM mode and tandem mass spectrometry. During the validation studies the HILIC-HPLC-MS method showed linear relation between AS-concentration and measured value over a wide range. Linearity was tested with the Mandel test revealing better fit by quadratic regression for three of 21 AS (NAcCys, NAcTyr and Pro). Recovery studies showed an influence of the matrix of the infusion solution, which led to deviations in the quotients areaAS / areaIS. As a result, quantitation by calibration with pure AS standard solutions was not possible within the given limits. Thus, validation was performed with matrix solution. Thereby it was shown that with the developed HPLC-MS method AS could be deter-mined in infusion solutions with high precision and accuracy. Nine of 21 AS were quantified in the range of 30%-350%, ten AS in the range of 50%-350% within the given requirements of pharmaceutical quality control (individual recovery value: 98% -102.0%, mean recovery value (threefold determination): 98.5% – 101.5%). His and Phe could not be quantified within the acceptance criteria; the reason for this would have to be found out in further studies. With the developed method the simultaneous quantitation of different AS infusion solutions, which differ in analytes concentrations with constant matrix concentration, can be realized. For instance, the formulations „Aminoplasmal® E 5% / 10% /15%“ can be mentioned, which could be determined by using the validated method. Sample preparation is fast and simple, consisting in addition of IS-formulation to the infusion solution and a single dilution step. Quantitation is performed by external standard calibration; the 5-point-regression-line is made by analyzing AS- and IS-standard solution after adding the electrolyte matrix solution, which is easily to produce. HPLC-MS analysis time is 35 min (including equilibration time), thus considerably shorter than the approximately 120 min required with the still common AS analytical method (ion exchange chromatography with postcolumn ninhydrin derivatization).

Im Rahmen der vorliegenden Arbeit wurden analytische Methoden zur Bestimmung des Verunreinigungsprofils von Erythromycin entwickelt, die der bestehenden Ph.Eur.-Methode überlegen sind. Die neue HPLC-Methode ist in der Lage, alle verwandten Verbindungen mit angemessener Präzision nachzuweisen und zu quantifizieren. Mit Hilfe der Massenspektrometrie konnten alle Hauptkomponenten und verwandten Verbindungen der Base, ihrer Ester und Salze eindeutig identifiziert und quantifiziert werden. Zudem konnten zwei neue verwandte Verbindungen von Erythromycin gefunden und als N,N-Didemethylerythromycin A und Anhydroerythromycin F identifiziert werden. / Within this work analytical methods for determination of the impurity profile of erythromycin were developed. These methods show clear improvements compared to the current methods described in the Ph.Eur. The new HPLC method is able to separate and quantify all related substances with an acceptable precision. The mass spectrometry shows the suitability for the direct qualitative and quantitative analysis of the base, ester and salts of erythromycin. Moreover using ion trap two new, unexpected EA related substances could be detected and identified as N,N-didemethylerythromycin A and anhydroerythromycin F.

This diploma thesis deals with hyphenation of liquid chromatography with mass spectrometric detection based on microfluidic chip. Firstly, a miniaturized ion source for atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photoionization (APPI) was constructed. The main component of this source was a glass microfluidic chip. Geometry and the working conditions of the chip were optimized. Since both ion sources work under the same conditions, possible advantages resulting from APCI/APPI combination were investigated. Furthermore, the performance characteristics of the sources were evaluated, and compared to the conventional high flow-rate sources. The best performing source, APCI, was then hyphenated with HPLC using low flow-rate. A method for separation of fatty acids methyl esters using Supelco 37 standard was developed. The separation conditions were as follows: C18 reversed stationary phase, and acetonitrile containing 0.1 % formic acids was used as the mobile phase. A temperature gradient was used in order to enhance the speed of the separation. The limits of detection and quantitation of for selected analytes using the chip micro-APCI were calculated, and compared to the ones obtained using a commercially available micro-APCI source. The method was used for separation of...

Micropartículas de quitosana contendo DMAE foram preparadas utilizando o método de coacervação simples. A morfologia das partículas foi observada utilizando um Microscópio Eletrônico de Varredura. O tamanho das partículas foi medido por técnica de espalhamento de luz. As partículas obtidas possuem forma esférica e superfície irregular. Apresentaram uma distribuição de tamanho entre 419 e 528 nm. O pequeno índice de polidispersividade sugere que a distribuição do tamanho de partícula é homogêneo. O rendimento do processo e eficiência de encapsulação foram de 90% e 63%, respectivamente. O desenvolvimento de um novo sistema de liberação requer uma metodologia analítica para a identificação e quantificação do fármaco. Neste trabalho um método simples por cromatografia de fase reversa desenvolvido para a análise do DMAE é apresentado. O DMAE foi analisado utilizando- se uma coluna Merck RP 18 column 5 m (125 x 4 mm D.I.). A fase móvel foi tampão fosfato pH 7,4; acetonitrila (99,5:0,5 v/v) a um fluxo de 0,5 mL.min-1. O comprimento de onda de detecção foi de 208nm à temperatura ambiente (25oC). Linearidade foi obtida para uma faixa de 1,5x10-4 a 6,0x10-4 mol.L-1. Para os ensaios intra e inter dia o coeficiente de variação foi menor que 10%. Este novo método desenvolvido para a quantificação do DMAE apresentou sensibilidade e seletividade, demonstrando ser um método vantajoso e confiável para a realização dos estudos propostos. O método de encapsulação mostrou-se adequado para a preparação de micropartículas de quitosana contendo DMAE, porque apresentou alto rendimento e excelente eficiência de encapsulação. / Chitosan microparticles containing DMAE were prepared by using the simple coacervation method. A scanning electron microscopy (SEM) was used to observe microparticles morphology. Particle size was measured by laser light scattering. Particles presented spherical shape, irregular surface and size distribution between 419 and 528 nm. The small polydispersity index suggested that size distribution is homogeneous. Process yield and encapsulation efficiency were 90% and 63%, respectively. The development of a new drug delivery system requires analytical methods for identification and quantification of this drug. In the present study a simple reversed-phase high performance liquid chromatography developed for DMAE assay is presented. The DMAE was analyzed using/by means of a 5 ?m Merck RP 18 column (125 x 4 mm I.D.). The mobile phase was phosphate buffer pH 7,4; acetonitrile (99,5:0,5 v/v) at a flow rate of 0,5 mL.min-1. Detection was carried out at 208nm at room temperature (25oC). Linearity was obtained from 1,5x10-4 to 6,0x10-4 mol.L-1. Intra and inter- assay coefficient of variation was less than 10 %. This new method developed to assay DMAE presented sensibility and selectivity, providing a useful and reliable means to perform the proposed studies. The encapsulation method was proven suitable to the preparation of chitosan microparticles containing DMAE because it presented high yields and excellent encapsulation efficiency.

No presente trabalho estudou-se por métodos analíticos (CCD e CLAE/UV) a atividade antioxidante dos flavonóides presentes na cana-de-açúcar (Saccharum officinarum L.), visando sua possível utilização como alimento funcional. As técnicas CLAE/UV e microfracionamento por CLAE permitiram o fracionamento e o isolamento dos flavonóides, os quais foram analisados por CCD utilizando reagentes específicos para detecção de substâncias antioxidantes. Os resultados obtidos permitiram identificar os flavonóides da cana-de-açúcar (folhas e garapa) com atividade antioxidante, através da comparação dos espectros de UV/DAD e tempo de retenção: orientina-O-ramnosídeo nas folhas e schaftosídeo, isoschaftosídeo, diosmina-8-C-glicosídeo, orientina e 4\',5\'-di-O-metil-luteolina-8-C-glicosídeo na garapa. Estes resultados justificam estudos nutricionais e/ou farmacológicos mais aprofundados a fim de classificar (ou não) a cana-de-açúcar como alimento funcional. / In the present study, analytical methods (TLC and HPLC/UV) were used to evaluate the antioxidant activity of the flavonoids of sugar cane (Saccharum officinarum L.) regarding to its possible use as a functional food. HPLC/UV and HPLC microfractionation techniques allowed fractionation and isolation of the flavonoids, which were evaluated by TLC using specific reagents to detect the antioxidant compounds. The results allowed to identify the flavonoids of sugar cane with antioxidant activity, based on comparison of UV-DAD spectra and retention time: orientin-O-rhamnoside in the leaves and schaftoside, isoschaftoside, diosmetin-8-C-glycoside, orientin and 4\'-5\'-dimethyl-luteolin-8-C-glycoside in the juice. Theses results justify more detailed nutricional and pharmacologic studies to classify (or not) sugar cane as a functional food.

Methods for polyamine derivatization with a panel of extrinsic fluorophores followed by HPLC with fluorescence and UV absorption detection have been developed. Four fluorophores were examined using polyamines and aminoglycosides. o-Phthalaldehyde (OPA) and fluorescamine are selective fluorophores that only react with primary amines; 9- fluorenylmethyl chloroformate (FMOC Cl) and dansyl chloride react with both primary and secondary amines. Reaction and HPLC conditions were optimized with each of the above fluorophores using a series of model mono- and diamines and then applied to natural and semi-synthetic polyamines. The amines that have been investigated are natural di- and polyamines: putrescine, cadaverine, spermidine, spermine, thermospermine, aminoglycosides: kanamycin, paramomycin, neomycin, and synthetic polyamine conjugates e.g. N⁴,N⁹-dioleoylspermine, N¹-cholesteryl spermine carbamate. The resultant derivatives were confirmed by off-line high resolution electrospray ionization mass spectrometry (HR ESI MS). The results show that the synthesis of polyamine derivatives in quantitative yield depends on the time of reaction, the temperature and the ratio of fluorophore reagent. Linearity of derivatization was calculated and regression coefficients ranged from 0.968 to 0.999 with good reproducibility. HR ESI MS analysis of the reaction products demonstrated complete derivatization of both primary and secondary amino groups with dansyl and FMOC fluorescence derivatives and of primary amine groups for OPA and fluorescamine derivatives. Under the ionization conditions used the dansyl derivatives showed, in addition to monovalent ions [M+H]⁺, divalent cations [M+2H]²⁺ because this chromophore contains a basic amine that can be easily protonated. FMOC derivatives gave prominent [M+Na]⁺ ions. The OPA derivatization reaction is rapid, but the products have poor stability. The derivatization with fluorescamine gave multiple products with glucosamine due to the presence of a chiral centre in the fluorophore. The relative quantum yields of the polyaminefluorophore derivatives were examined to determine the effect of intramolecular fluorescence quenching. Dansylation is the fluorescent derivatization method of choice.

Objetivos: O objetivo deste estudo foi avaliar a influência do Diabetes Mellitus do tipo 2 na farmacocinética da glimepirida em ratos Wistar e descrever o perfil através de modelo farmacocinética populacional (popPK). Metodologia: Os experimentos com animais foram aprovados pelo CEUA/UFRGS (protocolo #27892). O diabetes foi induzido com administração intraperitoneal de 100 mg/kg de nicotinamida, 15 minutos antes da administração intravenosa de 65 mg/kg de STZ. Os animais com nível de glicemia > 250 mg/dL foram considerados diabéticos. A glimepirida foi administrada na dose de 5 mg/kg via i.v. nos animais sadios (n = 11) e diabéticos (n = 9) e quantificada por CLAE-UV. A ligação às proteínas plasmáticas foi determinada por método de ultracentrifugação (Centrifree®). A análise farmacocinética não compartimental (software Phoenix®) foi realizada, assim como a modelagem farmacocinética populacional (software Monolix ®). Resultados e Discussão: A metodologia analítica para quantificação da glimepirida em plasma foi desenvolvida e validada, seguindo os critérios do FDA, apresentou sensibilidade, exatidão e precisão. O modelo de indução da diabetes produziu glicemia > 250 mg/dL. A ligação às proteínas plasmáticas não foi afetada pela doença (LPPSaudáveis = 99,3 ± 0,09%, LPPDiabéticos = 99,13 ± 0,075%, p > 0,05). O modelo farmacocinético populacional estrutural de 2 compartimentos com eliminação de primeira-ordem com covariável categórica (diabetes), foi usado para descrever os perfis plasmáticos de concentração-tempo da glimepirida após administração intravenosa na dose de 5 mg/kg a ratos saudáveis e diabéticos. O CL e a ASC0-inf dos animais diabéticos foram estatisticamente diferentes dos animais saudáveis, CLpop Saudáveis= 0,066 L/h para CLpop diabéticos = 0,024 L/h e ASCpop saudáveis = 19,24 μg/mL.h para ASCpop diabéticos = 59,64 μg.h/mL, indicando que a eliminação foi alterada nos animais diabéticos induzidos STZ. Conclusões: A modela gempossibilitou identificação do parâmetro que atribuiu variabilidade entre os grupos. Desta forma, a variabilidade interindividual foi quantificada e incluída no modelo. O modelo popPK final, nos permitiu elucidar os fatores que afetam a farmacocinética da glimepirida e prever mudanças na exposição em uma população específica. / Objective: The aim of this study was to evaluate the influence of diabetes mellitus type 2 on the pharmacokinetics of glimepiride in rats and describe the profile in population pharmacokinetic model (popPK). Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number). The diabetes was induced by intraperitoneal administration of NA (100 mg/kg) dissolved in saline 15 min before an intravenous administration of 65 mg/kg STZ in citrate buffer (pH 4.5) to overnight fasted rats. Animals with blood glucose level> 250 mg/dL were considered diabetic. After administered of glimepiride at a dose of 5 mg/kg i.v. bolus in healthy (n = 11) and diabetic animals (n = 9). Method HPLC-UV developed and validated quantified plasma concentrations. The plasma protein binding was determined by method ultracentrifugation (Centrifree®). Noncompartmental analysis of pharmacokinetic in Phoenix® software was performed, as well as the model pharmacokinetic population using Monolix®. Performed by Student's t-test for SigmaStat® software. Results and Discussion: The HPLC-UV method for quantification of glimepiride in plasma was developed and validated following requirements by FDA showing sensitivity, accuracy and precision. Induced diabetes model produced glucose> 250 mg / dL. The plasma protein binding was not affect by the disease (LPPSaudáveis = 99.3 ± 0.09%, LPPDiabéticos = 99.13 ± 0.075%, p> 0.05). The model pharmacokinetic population 2 compartments with eliminating first-order with categorical covariates diabetic was used to describe the plasma profile concentration-time glimepiride. The CL and AUC 0-inf of diabetic animals were significantly different. In healthy animals was Clpop Healthy = 0.066 L/h for diabetics Clpop = 0.024 L/h and healthy ASCpop = 19.24 g/mL.h ASCpop for diabetics = 59.64 g/mL.h, indicating that elimination was decreased in induced diabetic rats STZ. Conclusions: popPk enabled identification of the parameter assigned variability between the groups. Thus, the inter subject variability was measured and included in the model. The PBPK final model, allowed us to elucidate the factors that affect the pharmacokinetics of glimepiride and predict changes in exposure in a specific population.

Neste trabalho, foram desenvolvidos e validados métodos analítico e microbiológico, bem como estudo preliminar de estabilidade, cinética de degradação e citotoxicidade da Ceftarolina Fosamila em pó para solução injetável, um antibiótico da classe das cefalosporinas de quinta geração, indicado para pneumonias adquiridas na comunidade e infecções graves, de pele e tecidos moles. A validação do ensaio microbiológico pelo método de difusão em ágar cilindros em placa, delineamento 3x3, apresentou resultados satisfatórios, como especificidade, linearidade na faixa de 2,0 - 8,0 μg/mL, precisão (109,42 %), exatidão (102,3 %) e robustez. Soluções de Cefatarolina Fosamila do produto acabado expostas à radiação UVC (254 nm) e à degradação térmica a 60 °C foram utilizadas para avaliar a especificidade do bioensaio. A robustez foi avaliada através da alteração da concentração do meio inoculado (0,8 e 1,2 %). O desenvolvimento e validação de método por CLAE foi avaliado através da especificidade, linearidade, precisão, exatidão e robustez. No método cromatográfico foi utilizado cromatógrafo à liquido de alta eficiência SHIMADZU com coluna Agilent® C18, fase móvel (água com trietilamina 1,0% pH 5,0:acetonitrila 87:13 v/v). O método apresentou-se específico, linear, no intervalo de 5,0 - 60,0 μg/mL, preciso (110,0 %), exato (100,68 %) e robusto. Os métodos microbiológico e cromatográfico validados foram comparados estatisticamente e verificou-se não haver diferença significativa entre eles quando comparados através do teste “t” de Student. No estudo preliminar de estabilidade constatou-se ser estável em hidrólise ácida (0,1 M) e luz UVA no período avaliado, e instável frente à degradação térmica (40 e 60 °C), oxidativa com peróxido de hidrogênio, básica em NaOH (0,1 M e 0,01 M) e luz UVC. As cinéticas de degradação frente à luz UVC e degradação térmica 60 °C mostraram que as amostras possuem cinética de degradação de ordem zero e de segunda ordem, respectivamente. O ensaio de citotoxicidade demonstrou não haver diferença entre a condição normal e a amostra submetida à degradação forçada, sugerindo que os possíveis produtos de degradação formados não alteraram o resultado. / In this work, analytical and microbiological methods were developed and validated, as well as a preliminary study of the stability, degradation kinetics and cytotoxicity to Ceftaroline Fosamil powder for injectable solution, this is a fifth generation cephalosporin antibiotic indicated for community-acquired pneumonia and severe infections of the skin and soft tissues. The validation of the microbial assay by diffusion method in 3x3 cylinder agar delineated showed satisfactory results in specificity, linearity in the range of 2.0 - 8.0 μg / mL, precision (109.42 %), accuracy (102.3 %) and robustness. The development and validation of the method by HPLC was evaluated through specificity, linearity, precision, accuracy and robustness. In the chromatographic method was used high performance liquid chromatograph from SHIMADZU with Agilent® C18 column, mobile phase (water with triethylamine 1.0 % pH 5.0: acetonitrile 87:13 v/v). The method was linear, specific in the range of 5.0 - 60.0 μg/mL, accurate (110.0 %), exact (100.68 %) and robust. The validated microbiological and chromatographic methods were compared statistically and there was no significant difference between them when compared through Student's t-test. In the preliminary stability study, it was found stable in acid hydrolysis (0.1M) and UVA light in the period evaluated, and instable against thermal degradation (40 and 60 °C), oxidative with hydrogen peroxide, basic in NaOH (0.1 M and 0.0 1M) and UVC light. Samples exposed in UVC light an thermal degradation at 60°C showed degradation kinetics following zero order and second order, respectively. The cytotoxicity assay showed no difference between the normal condition and the sample submitted to forced degradation, suggesting that the possible degradation products formed did not change the result.

No description available.

Im Rahmen dieser Arbeit wurden sehr einfache, flüssigchromatographische Methoden zur Qualitätsanalytik gebräuchlicher Antimalaria-Medikamente (Amodiaquin, Mefloquin, Proguanil sowie die Kombination Artemether/Lumefantrin) entwickelt, die nur wenige, günstig erhältliche Chemikalien (Phosphatpuffer, Methanol) sowie gewöhnliche, kommerzielle RP-18-Säulen benötigen. Sie sind insbesondere zur Anwendung in Laboratorien in Entwicklungsländern geeignet und erfordern keine komplexen HPLC-Instrumente wie beispielsweise Gradientenpumpen oder Säulenthermostate. Der Verzicht auf Ionenpaarreagenzien ermöglicht es, dass eine stationäre Phase für mehr als nur einen einzigen Einsatzzweck verwendet werden kann und dass langwierige Äquilibrier- bzw. Spülschritte nicht notwendig sind. Alle Methoden arbeiten im isokratischen Elutionsmodus und durch die Verwendung kurzer Säulen (125 mm) konnten die jeweiligen Analysenzeiten zusätzlich verringert werden. Hierdurch ist zudem eine Reduzierung des Fließmittelverbrauches möglich. Während der Methodenentwicklung wurden charakteristische, aus dem Herstellungsweg des jeweiligen Arzneistoffes stammende potentielle Verunreinigungen berücksichtigt. Ihre Bestimmung erlaubt eine Aussage über die Herkunft eines Wirkstoffes bzw. eines Arzneimittels, da das Verunreinigungsmuster einer Substanz oftmals die Zuordnung zu einem bestimmten Herstellungs- bzw. Reinigungsprozess ermöglicht. Alle Methoden wurden hinsichtlich der Linearität innerhalb des Arbeitsbereiches sowie der Wiederholpräzision charakterisiert. Es wurde eine gute Reproduzierbarkeit gefunden. Die Nachweis- und Bestimmungsgrenzen der untersuchten Verunreinigungen lagen bei einem Level von je 0.1 %. Durch gezielte Variation wurde der Einfluss wechselnder Trenntemperaturen sowie schwankender pH-Werte der jeweiligen mobilen Phase und die hieraus resultierenden Effekte untersucht. Hierbei zeigte sich, dass die Methoden sehr robust gegenüber diesen Einflussgrößen sind und somit für die Anwendung mit einfach ausgestatteten HPLC-Systemen sowie besonders für den Einsatz in tropische Gebieten mit wechselnden klimatischen Bedingungen gut geeignet sind. Flüssigchromatographische Methoden spielen heute in der pharmazeutischen Analytik vor allem zur Bestimmung der Reinheit eines Arzneistoffes eine herausragende Rolle und sind in nahezu jeder Monographie der wichtigsten Arzneibücher (z. B. im Ph. Eur.) zu finden. Einfach durch-führbare Untersuchungsmethoden, wie beispielsweise die im GPHF-Minilab® angewandte Dünnschichtchromatographie, erfordern im Vergleich zur HPLC weniger komplexe und teure Instrumente und können selbst in entlegenen Gebieten ohne Laboratorium durchführt werden. Sie verfügen allerdings über eine nur sehr geringe Genauigkeit und Reproduzierbarkeit, da sowohl die praktische Durchführung als auch die anschließende Auswertung rein manuell bzw. visuell erfolgt und somit in hohem Maße einer Beeinflussung durch den jeweiligen Analytiker unterworfen ist. Die entwickelten HPLC-Methoden wurden mit dünnschichtchromatographischen Verfahren verglichen, hierbei besonders unter dem Aspekt der visuellen und der instrumentellen Auswertung der Chromatogramme zur Bestimmung des Gehaltes einer unbekannten Probe. Hierbei konnte aufgezeigt werden, dass die Dünnschichtchromatographie der Flüssigchromatographie eindeutig unterlegen ist, insbesondere wenn die Auswertung nicht mittels eines entsprechenden Scanners sondern rein visuell erfolgt: Nur in den wenigsten Fällen ist es möglich, eine annähernd präzise Aussage über den Gehalt zu treffen und zudem ist die Bestimmung der Verwandten Substanzen nur sehr bedingt möglich. Durch den Einsatz von Auftragegeräten bzw. Plattenscannern kann die Genauigkeit zwar signifikant erhöht werden, allerdings sind solche Instrumente im Verhältnis wesentlich teurer als einfache, modulare HPLC-Systeme und zählen heute in den wenigsten Laboratorien zum Standardinventar. Vereinfachte chromatographische Methoden können ein wichtiges Hilfsmittel für Kontrolllaboratorien in Entwicklungsländern sein, wenn komplexe, etablierte Protokolle nur eingeschränkt angewendet werden können. Durch die Kombination aus dünnschichtchromatographischer Basisanalytik und einer flächendeckenden Untersuchung mittels HPLC lässt sich die Arzneimittelqualität sehr gut überprüfen, die regulatorischen Organe eines Landes entsprechend zu entlasten und die Versorgung der Bevölkerung mit qualitativ einwandfreien Medikamenten zu gewährleisten. Ein weiterer Teil der Arbeit befasst sich mit der Stabilitätsanalytik individuell hergestellter, Noradrenalin-haltiger Injektionslösungen. Solche Rezepturen werden oftmals in Krankenhausapotheken im Rahmen der Defektur auf Vorrat durch Verdünnen der entsprechenden kommerzieller Fertigarzneimittel mit isotonischer Kochsalzlösung zubereitet, um z. B. für Notfallsituationen am Wochenende die Rezepturen vorrätig zu haben. Durch die Untersuchungen wurde geprüft, inwieweit der übliche Verdünnungsgrad von 0.1 % einen Einfluss auf die Stabilität des Noradrenalins hat und welche Lagerungsbedingungen für die Zubereitungen empfohlen werden können. Nach der Lagerung unter verschiedenen Bedingungen (gekühlt, bei Raumtemperatur sowie jeweils mit bzw. ohne Lichtschutz) konnte gezeigt werden, dass die Gehalte an Noradrenalin bei keiner der untersuchten Lagerungsbedingungen unter einen Wert von 99.0 % fielen. Individuell hergestellte Noradrenalin-Injektionslösungen können somit bis zu sieben Tage im Voraus hergestellt und für die Anwendung am Patienten bereit gehalten werden. Die Lösungen sollten dennoch gekühlt und unter Lichtschutz aufbewahrt werden, um den Abbau des Arzneistoffes und eine mikrobielle Kontamination zu minimieren. / This work focuses on the development of simple, liquid chromatographic methods for the quality analysis of common antimalarial medicines, i.e. amodiaquine, mefloquine, proguanil and the fix-dose combination of artemether and lumefantrine. They require a minimum of readily available, cheap chemicals (e.g. phosphate buffers or methanol) and commercially available RP-18 columns. They were designed for use in quality control laboratories in resource-restraint environments, e.g. in laboratories in the developing world, and do not require complex HPLC instrumentation which are either not available or affordable, e.g. with expensive gradient pumps or column ovens. Ion-pairing reagents were strictly avoided during method development which is a great advantage for routine application: long equilibration and flushing procedures are not necessary and a column can be used for more than one single method. An isocratic elution mode was applied and using short analytical columns (125 mm) allows reducing the analysis time and eluent consumption, respectively, to a minimum. During method development impurities being characteristic for the respective synthesis pathway(s) of the active substances were considered. Thus, determining the quality of a com-pound with regard to its content and purity is possible and distinct manufacturers or sources can be identified. Linearity and repeatability were assessed and a good reproducibility was found. Limits of detection and quantitation, respectively, were measured and the respective impurities can be determined to a level of 0.1 %. By varying the separation temperature and the pH-value of the respective mobile phases method ruggedness was investigated. A high grade of robustness towards these parameters could be confirmed, indicating that the methods are suitable for being used in tropical environments. Liquid chromatographic methods play an important role in pharmaceutical analysis today and they are widely applied for determining the purity of a compound. Respective protocols have been added to almost every monograph of the most important pharmacopoeias, e.g. the European Pharmacopoeia. Of note, those protocols may not be applied in every laboratory without limitations. Although very simple methods of analysis, e.g. thin-layer chromatography which is described in the manuals of the GPHF-Minilab®, require less expensive instruments and may even be applied in resource-restraint environments, they exhibit a poor accuracy and reproducibility. Preparing and evaluating the plates manually may strongly bias the results, and in addition this highly depends from the respective analyst who performs the tests. The comparison of thin-layer chromatographic assays applying a manual and an instrumental evaluation to results obtained from liquid chromatographic tests clearly exhibited the disadvantages particularly for determining the quality of a compound. It is almost impossible to exactly determine the content of a sample, and a comprehensive test for related substances cannot be carried out. Using application devices and plate readers may increase the accuracy, however such instruments are a lot more expensive than simple, modular HPLC systems and normally do not belong to the standard inventory of a quality control laboratory. Simplified HPLC methods may serve as helpful instruments for the regulatory infrastructure of developing countries. Combining them with basic thin-layer chromatographic tests and applying them comprehensively may enable the respective quality control laboratories to ensure the nationwide supply with immaculate medicines. In the second part of this work the stability of individually prepared dilutions of commercially available norepinephrine injectable solutions was investigated and recommendations for storage conditions were derived. In hospital pharmacies it is a common practice to prepare such solutions from proprietary products by diluting them with isotonic sodium chloride solution or other suitable buffer solutions, respectively, to a concentration of 0.01 mg/ml (1:100). This is important particularly in case of emergencies, e.g. on the weekend or during holidays, because the respective medication can be held in stock even when the pharmacy is closed. Within the framework of the experiments the influence on the stability of norepinephrine after diluting the respective proprietary product with sodium chloride solution was investigated. The samples were stored with and without refrigeration, and unprotected and protected from sunlight. The results showed that under none of the investigated storing conditions the content of norepineprhine decreased significantly. The lowest content which was found was 99.0 %. Thus, individual norepinephrine injectable solutions can be prepared in advance and storing them is possible for at least seven days. Although a degradation of the active ingredient or a diffusion in the primary packaging material (e.g. a syringe made from polyethylene) could not be observed, it is recommended to store the preparations in the refrigerator and protected from light. A microbial contamination may also be avoided like this.