A análise de impurezas é uma etapa importante no controle de qualidade do insumo farmacêutico e do produto final. Provenientes da síntese do medicamento ou dos excipientes, mesmo em pequenas concentrações, as impurezas podem afetar a eficácia e a segurança. No presente trabalho foram desenvolvidos e validados dois métodos empregando cromatografia líquida de alta eficiência (CLAE) para a avaliação do besifloxacino e sua impureza de síntese, um com eluição isocrática e outro com eluição gradiente. As análises por CLAE com modo de eluição isocrática foram executadas utilizando coluna Ciano, mantida a 25 °C. A fase móvel foi constituída por 0,5% de trietilamina (pH 3,0) : acetonitrila (88:12 v/v), eluída na vazão de 1,0 mL/min com detecção a 330 nm. O método com eluição gradiente foi conduzido com a mesma coluna e componentes da fase móvel modificando apenas as proporções entre fase orgânica e aquosa durante as análises. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados dentro dos limites aceitáveis. Os métodos apresentaram-se lineares na faixa de 140 a 260 μg/mL para o besifloxacino e de 0,3 a 2,3 μg/mL para a impureza A. Com volume de injeção de 20 μL, os limites de detecção e quantificação foram, respectivamente, 0,07 e 0,3 μg/mL. A precisão foi alcançada para todas as análises realizadas, obtendo DPR inter-dia igual a 6,47 e 6,36 para a impureza A com eluição isocrática e gradiente, respectivamente. A exatidão foi superior a 99% e a robustez apresentou resultados satisfatórios. No método isocrático obteve-se tempo de análise de 25 min e no gradiente de 15 min. O número de pratos teóricos no modo isocrático foi na ordem de 5000 enquanto no modo gradiente foi na ordem de 45000, ou seja, obteve-se maior eficiência da coluna com alteração da composição da fase móvel durante a eluição. No insumo besifloxacino e no produto farmacêutico utilizados neste trabalho, impurezas relacionadas estavam presentes, mas a impureza A não foi detectada. Os métodos propostos, considerando-se o limite de quantificação, podem ser aplicados na determinação quantitativa da impureza A na análise da matéria prima do besifloxacino, assim como na suspensão oftálmica. / Analysis of impurities is an important step in quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. In the present study two chromatographic methods were developed and validated for high-performance liquid chromatography (HPLC) for the assessment of besifloxacin and its synthesis impurity, one with isocratic and another with gradient elution. Analyses by HPLC in isocratic elution mode were performed using Cyano column maintained at 25 °C. Mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 ml/min with detection at 330 nm. The method with gradient elution was carried out with the same column and mobile phase components modifying only proportion between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, obtaining results within acceptable limits. The methods presented a linear response from 140-260 μg/mL for besifloxacin and from 0.3 to 2.3 mg/mL for impurity. With the injection volume of 20 μL, the limit od detection and limit of quantitation were, respectively, 0.07 and 0.3 μg/mL. Precision was achieved for all analyses, obtaining inter-day RSD equal to 6.47 and 6.36 for impurity A with isocratic and gradient elution, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method was obtained analysis time 25 min and 15 min gradient. The number of theoretical plates in the isocratic mode was of the order of 5000 while in the gradient mode was of the order of 45000, that is, gave greater efficiency of the column by changing the mobile phase composition during elution. In raw material of besifloxacin and pharmaceutical product used in this study, related impurities were present but the impurity A was not detected. The proposed methods, considering the limit of quantitation, can be in quantitative determination of impurity A in the analysis of besifloxacin raw material, as well as in ophthalmic suspension.

A vigabatrina (VGB) é um fármaco anticonvulsivante que apresenta apenas a forma farmacêutica sólida disponível para uso. Na área hospitalar, devido à ausência de medicamentos na forma farmacêutica líquida, são preparadas derivações a partir de comprimidos e cápsulas para adequar a administração da dose prescrita. No entanto, a falta de estudos de estabilidade podem comprometer a eficácia e segurança destas derivações. O objetivo deste trabalho foi analisar a estabilidade química de derivações de comprimidos de vigabatrina em condições de armazenamento sob diferentes temperaturas e variações na embalagem utilizada. A análise das derivações de VGB foi realizada através de cromatografia líquida de alta eficiência (CLAE). O método descrito na Farmacopeia Britânica (2016) foi covalidado quanto a especificidade, linearidade, precisão e exatidão. Amostras de derivação de VGB foram preparadas em triplicata e acondicionadas em frascos de vidro e de PET âmbar. Após, foram armazenadas sob três diferentes condições de temperatura: temperatura ambiente (15 a 30 °C), sob refrigeração (2 a 8 °C) e em estufa (40 °C). Foram coletadas amostras armazenadas nas diferentes embalagens a cada 7 dias, por um período de 35 dias para as amostras conservadas em temperatura ambiente e refrigerada. O mesmo procedimento foi realizado para as amostras conservadas em estufa, porém por um período de 28 dias Também foi analisado o pH das amostras em cada tempo de coleta. As derivações de VGB foram analisadas por CLAE e apresentaram variação dentro dos limites preconizados pela Farmacopeia Britânica 2016, até 21 dias para frascos de vidro e de PET âmbar para as temperaturas ambiente e refrigerada. As amostras de VGB conservadas em estufa, apresentaram redução acima de 10% após 7 dias de estudo. A menor variação de pH ocorreu em frasco de vidro âmbar armazenado sob refrigeração. O resultado deste estudo serve de referência no preparo de derivações de VGB para uso hospitalar, pois apresentou intervalo de tempo confiável e condições de armazenamento adequadas para sua utilização. Desta forma, os pacientes pediátricos que utilizam doses fracionadas ou pacientes em uso de sondas nasogástricas terão as derivações adequadamente preparadas, reduzindo o risco de erros de diluição e contaminação microbiológica, melhorando a eficácia e segurança terapêutica. / Vigabatrin (VGB) is an anticonvulsant drug that has only solid dosage form available for use. In hospital, due to lack of medicines in liquid dosage form, extemporaneous preparations are prepared from tablets and capsules to adapt the administration of the prescribed dose. However, the lack of stability studies may compromise the efficacy and safety of these preparations. The aim of this study was to analyze the chemical stability of VGB extemporaneous preparation from tablets at storage conditions in different temperatures and variations of packaging used. The analysis of VGB extemporaneous preparations were performed by high-performance liquid chromatography (HPLC). The method described in British Pharmacopoeia (2016) was co-validated for specificity, linearity, precision and accuracy. VGB extemporaneous preparations were prepared in triplicate and placed in amber glass and PET bottles, which were stored under three different conditions: at room temperature (15 to 30 °C), under refrigeration (2 to 8 °C), and oven (40 °C). Samples of preparations stored at room temperature and refrigeration were collected every 7 days along 35 days. The same was done for solutions kept at 40 °C, but for a period of 28 days. It was also analyzed the preparations pH for each sampling time VGB extemporaneous preparations were analyzed by HPLC and demonstrated variations within the limits of British Pharmacopoeia (2016) up to 21 days in amber glass and PET bottles at room and refrigerated temperatures. VGB content for preparations kept in oven decreased above 10% after 7 days of study. The lowest pH change occurred in amber glass bottle stored under refrigeration. Results of this study can be applied as a reference for VGB extemporaneous preparation in hospital, once it was demonstrated the reliability of storage time interval and proper conditions for the use. Thus, pediatric patients with fractionated doses or patients using nasogastric probe will have adequately prepared extemporaneous preparations, reducing the risk of dilution errors and microbiological contamination.

The dynamic S-palmitoylation regulates many intracellular events, including protein trafficking, anchoring, targeting, and protein-protein interactions. Direct detection of S-palmitoylation by conventional liquid chromatography-mass spectrometry (LC-MS) methods is challenging because of the tendency of palmitoyl loss during sample preparation and gas phase fragmentation. Additionally, the high hydrophobicity of the palmitoyl group can prevent proper elution of palmitoyl peptides from the commonly used C18 column. Here, we developed a comprehensive strategy tailored for S-palmitoyl detection using three palmitoyl peptide standards. We found that S-palmitoylation was largely preserved in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent and that various fragmentation methods provided complementary information for palmitoyl localization. Moreover, S-palmitoyl peptides were efficiently analyzed using a C4 column and the derivatization of free cysteine with a hydrophobic tag allowed relative quantification of palmitoyl peptides and their unmodified counterparts. We further discovered potential complications to S-palmitoylation analysis caused by the use of ProteaseMAXTM, an MS-compatible detergent. The hydrophobic degradation products of ProteaseMAXTM reacted with the free cysteine thiols, generating artifacts that mimic S-acylation and hydroxyfarnesylation. Another MS-compatible detergent, RapiGestTM, did not produce such artifacts, and showed the ability to stabilize S-palmitoylation by preventing thioester hydrolysis and dithiothreitol-induced thioester cleavage. Moreover, we found that the palmitoyl peptide GCpalmLGNAK could undergo intermolecular palmitoyl migration from the cysteine to the peptide N-terminus or the lysine side chain during sample preparation, and this could lead to false discovery of N-palmitoylation. RapiGestTM inhibited such migration, and is thus recommended for S-palmitoyl sample preparation. We then applied the established method to analyze the regulator of G-protein signaling 4 (RGS4) which had been reported to undergo S-palmitoylation by radioactive labeling. It had also been reported that the S-palmitoylation state of RGS4 affects its GTPase activity. With LC-MS/MS analysis, we found that the addition of palmitate to the cell culture medium in metabolic labeling experiments could boost the level of S-palmitoylation, leading to false discovery of new S-palmitoylation site(s). We also noted discrepancies between the S-palmitoylation sites identified by radioactive labeling and by LC-MS/MS analysis. Further studies are needed to evaluate the reliability of S-palmitoyl detection by these two methods.

Propolis is an approved food additive containing flavonoids as a major active constituent. Variability has been found in the composition of propolis in distinctive regions and it was noticed that there are limitations in the analysis of propolis. In this study, the identification of ten flavonoids and residual antibiotics in propolis was investigated by using several liquid chromatography techniques, including reversed-phase high-performance liquid chromatography (RP-HPLC), microemulsion LC (MELC) and ultra-performance LC (UPLC). The ten flavonoids that were selected for this research include rutin, myricetin, quercetin, apigenin, kaempferol, pinocembrin, CAPE, chrysin, galangin and acacetin while chlortetracycline, oxytetracycline and doxycycline were selected to examine the residual antibiotics in propolis. For the analysis of the selected flavonoids, routine RP-HPLC method was found to be the best method, while MELC technique was found more efficient for the analysis of the selected antibiotics. Solid phase extraction with HLB sorbent was utilised in the analysis of antibiotics for clean-up of propolis. In method development studies for flavonoids and antibiotics, one-factor-at-a-time (OFAT) approach was followed. The final optimised method for the analysis of flavonoids as well as the method. for the analysis of antibiotics was validated using the ICH guidelines, and various aspects, such as the linearity, selectivity, accuracy, recovery, robustness and stability parameters, were examined. Development of efficient conventional method for the extraction of flavonoids from propolis was studied extensively in the present research work using different extraction techniques such as maceration, hot extraction, ultrasound assisted extraction. Among all extraction experiments, ethanolic extraction using ultrasound extraction method was the best efficient approach. This thesis shows that, in general, the performance of O/W MELC is superior to that of conventional HPLC for the determination of residual antibiotics in propolis. UPLC was not suitable for the analysis of flavonoids and antibiotics. The conventional LC was the only technique to separate the ten flavonoids but MELC was able to separate nine of the flavonoids with faster analysis time. This work also showed that MELC uses cheaper solvents. This considerable saving in both cost and time will potentially improve efficiency within quality control.

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 hidroxicloroquina (HCQ) é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas são estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que o metabolismo estereosseletivo parece ser função da espécie estudada, o que implica na necessidade de métodos seletivos para a determinação de seus enantiômeros em materiais biológicos. Assim, propôs-se o desenvolvimento e a validação de métodos para análise dos enantiômeros do fármaco inalterado e de seus principais metabólitos em frações microssomais de homogeneizados de fígado de ratos e camundongos. Para tanto foram empregadas as técnicas de eletroforese capilar (CE) e de cromatografia líquida de alta eficiência (HPLC). Inicialmente foi desenvolvido um método por HPLC, em uma etapa, para a quantificação dos enantiômeros de dois metabólitos da HCQ, desetilcloroquina (DCQ) e desetilhidroxicloroquina (DHCQ) em microssomas de fígado de ratos e camundongos. A separação foi efetuada utilizando-se a coluna Chiralpak AD-RH e hexano:isopropanol (92:8, v/v) acrescido de 0,1% de dietilamina como fase móvel. O procedimento de extração líquido-líquido foi utilizado para a preparação das amostras. A metodologia desenvolvida resultou na completa resolução dos enantiômeros da HCQ, DCQ e DHCQ e pode ser considerada adequada, visto que os parâmetros de validação mostraram valores dentro dos limites exigidos na literatura. O segundo método desenvolvido permitiu a quantificação dos enantiômeros dos três metabólitos da HCQ: DCQ, DHCQ e bisdesetilcloroquina (BDCQ) em microssomas de fígado de camundongos. A separação foi efetuada utilizando-se um tubo capilar de sílica fundida e solução do eletrólito tris(hidroximetil)aminometano 100 mmol/L, ajustada a pH 9,0 com ácido fosfórico e acrescida de 1% (m/v) de β –CD - sulfatada e 30 mmol/L de β –CD - hidroxipropilada. O procedimento de extração líquido-líquido foi eficiente para remover interferentes e os parâmetros de validação mostraram valores dentro dos limites exigidos na literatura. Os métodos desenvolvidos foram aplicados no estudo in vitro do metabolismo da HCQ em frações microssomais de fígado dos animais, verificando-se que o principal metabólito formado é o (-)-(R)-DHCQ, para ambas espécies estudadas. / Hydroxychloroquine (HCQ) is an important chiral drug used specially in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, with stereoselective pharmacokinetic and pharmacodinamic properties. Concerning these properties, some previous studies indicate that the stereoselective metabolism seems to be a function of the studied species, therefore selective methods are required for the determination of its enantiomers in biological matrix. Thus, the present work reports the development and validation of methods for the analysis of the enantiomers of HCQ and its main metabolites in microsomal fraction of rats and mice liver homogenates. Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were used for this purpose. Initially, a one-step HPLC method was developed for the quantification of the enantiomers of two HCQ metabolites, desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) in microsomal fraction of rats and mice liver homogenates. The separation was performed on a Chiralpak AD-RH column using hexane:isopropanol (92:8, v/v) plus 0.1% diethylamine as the mobile phase. Liquid-liquid extraction procedure was used for sample preparation. The developed methodology resulted in the complete resolution of HCQ, DCQ and DHCQ enantiomers and can be considered suitable because the validation parameters are in accordance with the limits established in the literature. The second developed method allowed the quantification of the enantiomers of the three HCQ metabolites, DCQ, DHCQ and bisdesethylchloroquine (BDCQ) in microsomal fraction of mice liver homogenates. The separation was performed using a fused-silica capillary tube and tris (hydroxymethyl) aminometane 100 mmol/L electrolyte solution, adjusted at pH 9.0 with phosphoric acid, containing 1% (w/v) sulfated- β -CD and 30 mmol/L hydroxypropyl- β -CD. The liquid-liquid extraction procedure was efficient to remove interferents and the validation parameters showed values within accordance to the literature. The developed methods were applied in the in vitro metabolism study of HCQ in microsomal fractions of the liver of the animals and it was verified that the main metabolite formed is (-)-(R)-DHCQ for both animal species studied.

As teorias neurobiológicas defendem que a esquizofrenia é essencialmente causada por alterações bioquímicas e estruturais do cérebro. A importância que os aminoácidos tem com a fisiopatologia da esquizofrenia e, por este envolvimento encontrar-se pouco esclarecido é que esta pesquisa teve como propósito desenvolver e validar uma metodologia analítica para a análise simultânea dos aminoácidos: glutamato, aspartato, serina, glicina, arginina e lisina em plasma de pacientes com diagnóstico de esquizofrenia, utilizando para isto a cromatografia líquida de alta eficiência com detecção por fluorescência. Os analitos foram inicialmente extraídos através do processo de precipitação protéica, com o solvente orgânico acetonitrila. Para que pudessem ser detectados por fluorescência os aminoácidos foram derivatizados empregando orto-ftalaldeído na presença de B-mercaptoetanol. O tempo total de separação foi de 18 minutos em coluna analítica LiChroCART® RP-18 (Merck, 125mm, 4mm d.i., 5m) no modo de eluição gradiente com tampão acetato de sódio 0,1 mol/L (pH 7,0, A) e acetonitrila e água (B), na proporção 70:30, respectivamente, para posterior detecção por fluorescência (excitação 340 nm, emissão 450 nm). O método foi validado segundo os critérios estabelecidos pela Agência Nacional de Vigilância Sanitária (ANVISA). A resposta do detector encontrou-se linear na faixa de 9,6 a 192 pmol/mL para todos os aminoácidos. O limite de detecção estabelecido foi de 7,68 pmol/mL e o limite de quantificação foi de 9,6 pmol/mL. A recuperação foi superior a 70%. A precisão e exatidão intra-ensaio variou de 3,6% a 5,3% e 3,1% a 8,3%, respectivamente. A precisão e exatidão interensaio variou de 3,6% a 7,1% e 3,1% a 11,4%, respectivamente. A especificidade foi determinada para os seguintes interferentes: lorazepam, diazepam, clonazepam, alprazolam, haloperidol, levomepromazina, propranolol, ranitidina, fluoxetina, olanzapina, carbamazepina, diclofenaco, amiodarona, sulfametoxazol e, ainda, plasma hemolisado, normal e lipêmico. O método desenvolvido e validado mostrou ser eficiente na determinação simultânea de aminoácidos podendo ser aplicado em amostras de pacientes esquizofrênicos a fim de investigar seu envolvimento com esta desordem psiquiátrica tão intrigante. / Schizophrenia is primarily caused by structural and biochemical changes in the brain according to the main neurobiological theory, including the amino acids concentrations in the human plasma. The relation between these amino acids concentrations and the schizophrenia needs more clarification. Therefore, an analytical tool is necessary to provide which of these amino acids are related with this mental disease. The aim of this research was to develop and to validate an analytical methodology using High Performance Liquid Chromatography with fluorescence detection for simultaneous analysis of the main human amino acids, which are: glutamate, aspartate, serine, glycine, arginine and lysine in plasma of schizophrenic patients. Protein precipitation was the extraction technique chosen for the amino acids determination using acetonitrile as organic solvent. The derivatization was conducted by the reaction between the amino acids and ortho-phthalaldehyde in the presence of B-mercaptoethanol. The separation was achieved using the analytical column LiChroCART® RP-18 (Merck, 125mm, 4mm ID, 5mm) by gradient elution in 18 minutes. The mobile phase was composed by sodium acetate buffer 0.1 mol / L (pH 7.0; A) and acetonitrile: water (B) (70:30, v/v). The detection was performed by fluorescence (excitation 340 nm, emission 450 nm). The method was validated according to criteria established by the regulatory agency (ANVISA). The detector response was found linear in the range 9.6 to 192 pmol / mL for all amino acids. The detection limit was set at 7.68 pmol / mL and the limit of quantification was 9.6 pmol / mL. The recovery was above 70%. The precision and accuracy intra-assay ranged from 3.6% to 5.3% and 3.1% to 8.3%, respectively. The precision and accuracy inter-assay ranged from 3.6% to 7.1% and 3.1% to 11.4%, respectively. The specificity was determined for the following possible interferents: lorazepam, diazepam, clonazepam, alprazolam, haloperidol, levomepromazine, propranolol, ranitidine, fluoxetine, olanzapine, carbamazepine, diclofenac, amiodarone, trimethoprim. Plasma hemolyzed, lipemic and normal were evaluated. This method was adequate to simultaneous determination of amino acids, therefore it can be applied to samples of schizophrenic patients and consequently, providing information of the main amino acids involvement with this psychiatric disorder.

The bacterium Rhodococcus is a potential source for novel antimicrobial metabolites. Recently, the Rhodococcus strain MTM3W5.2 was isolated from a soil sample collected from Morristown, East Tennessee and was found to produce an inhibitor molecule that is active against similar Rhodococcus species. The aim of this research is to extract, purify, and characterize the active compound. The compound was obtained from both agar and broth cultures of strain MTM3W5.2 and purified by primary fractionation of crude extract on a Sephadex LH-20 column, followed by semi-preparative reversed phase column chromatography. Final purification was achieved using multiple rounds of an analytical C18 HPLC column. Based on the results obtained from UV-Vis, FT-IR, and HR-MS, the molecule is a polyketide with a molecular formula of C52H78O13 and an exact mass of 911.5490 amu. The partial structure of this compound has been determined using 1D and 2D NMR spectroscopy.

Due to the large gaps in the fossil record, the evolutionary history of the mammalian order Cetacea is incomplete and controversial. Increasingly researchers are utilizing molecular and biochemical procedures to supplement cetacean paleontology. One of these methods is the comparison of amino acid sequences of myoglobin among species of this order. since this method is time-consuming and expensive, an alternative procedure is desirable.

No description available.