RESUMO
Resveratrol, a modulator of several signaling proteins, can exert off-target effects involving the peroxisome proliferator-activated receptor (PPAR) transcription factors. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand-binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry allowed the binding affinities of resveratrol, resveratrol 3-O-glucuronide, resveratrol 4-O-glucuronide, and resveratrol 3-O-sulfate to both PPAR-LBDs to be determined. Interaction of resveratrol, resveratrol 3-O-glucuronide, and resveratrol 4-O-glucuronide with PPARγ-LBD occurred with binding affinities of 1.4, 1.1, and 0.8 µM, respectively, although only resveratrol bound to the PPARα-LBD with a binding affinity of 2.7 µM. Subsequently, X-ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ-LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ-LBD and resveratrol revealed the presence of one molecule of resveratrol bound to the LBD of PPARγ, with the ligand occupying a position close to that of other known PPARγ ligands. Transactivation assays were also performed in HepG2 cells, with the results showing that resveratrol was not a PPAR agonist but instead was able to displace rosiglitazone from PPARγ and Wy-14643 from PPARα with IC50 values of (27.4±1.8) µM and (31.7±2.5) µM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα.
Assuntos
Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Estilbenos/metabolismo , Estilbenos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Modelos Moleculares , Receptores Ativados por Proliferador de Peroxissomo/química , Pirimidinas/farmacologia , Resveratrol , Rosiglitazona , Relação Estrutura-Atividade , Tiazolidinedionas/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.
Assuntos
Cromatografia em Gel , PPAR alfa , PPAR gama , Extratos Vegetais , PPAR gama/metabolismo , PPAR gama/química , PPAR alfa/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ligantes , Espectrometria de Massas , Rosiglitazona/farmacologia , Rosiglitazona/química , Humanos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/análise , PirimidinasRESUMO
An integrated chromatographic system was developed to rapidly investigate the biocatalytic properties of ω-transaminases useful for the synthesis of chiral amines. ATA-117, an (R)-selective ω-transaminase was selected as a proof of concept. The enzyme was purified and covalently immobilized on an epoxy monolithic silica support to create an immobilized enzyme reactor (IMER). Reactor efficiency was evaluated in the conversion of a model substrate. The IMER was coupled through a switching valve to an achiral analytical column for separation and quantitation of the transamination products. The best conditions of the transaminase-catalyzed bioconversion were optimized by a design of experiments (DoE) approach. The production of (R)-1-(4-methoxyphenyl)propan-2-amine and (R)-1-methyl-3-phenylpropylamine, intermediates for the synthesis of the bronchodilator formoterol and the antihypertensive dilevalol respectively, was achieved in the presence of different amino donors. The enantiomeric excess (ee) was determined off-line by developing a derivatization procedure using Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide reagent. The most satisfactory conversion yields were 60% for (R)-1-(4-methoxyphenyl)propan-2-amine and 29% for (R)-1-methyl-3-phenylpropylamine, using isopropylamine as amino donor. The enantiomeric excess of the reactions were 84%R and 99%R, respectively.
Assuntos
Cromatografia/métodos , Enzimas Imobilizadas/química , Transaminases/química , Aminação/fisiologia , Aminas/química , Biocatálise , Catálise , Propilaminas/química , EstereoisomerismoRESUMO
The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.
Assuntos
Sistemas On-Line/instrumentação , Fosfoproteínas/química , Líquido Amniótico/química , Reatores Biológicos , Caseínas/química , Cromatografia Líquida/métodos , Feminino , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Fosfopeptídeos/isolamento & purificação , Fosfoproteínas/metabolismo , Gravidez , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
The review examines the most recent achievement of immobilized Penicillin G acylase (PGA) as chiral stationary phases (PGA-CSP) for the separation of acidic enantiomers. Particular attention is paid to the influence of structural variations of a large number of analytes on retention and enantioselectivity with a special emphasis on advances in the elucidation of the chiral recognition mechanism. Docking calculations and molecular dynamic simulations are discussed to rationalize the origins of enantioselective behaviour. The review also touches the chiral behaviour of PGA in partial filling CE. The CE results, obtained using PGA in free solution, suggest that the immobilization procedure used in the development of PGA-CSPs did not alter the enantioselective properties of the enzyme.
Assuntos
Cromatografia Líquida/métodos , Eletroforese Capilar/métodos , Penicilina Amidase/química , Sítios de Ligação , Simulação por Computador , Fenilacetatos/química , EstereoisomerismoRESUMO
In the present study, a single HPLC method was developed for the determination of glycine and threonine in cicatrizants. Two different preparations of a cream and an ointment, and the corresponding bandages, onto which the formulations were applied, were studied. The method involved matrix solubilisation with dichloromethane, liquid-liquid isolation of gly and thr with aqueous 1N NaOH, and derivatization with phenylisothiocyanate. Reversed-phase HPLC separation was carried out by gradient elution with 20mM aqueous NaClO4 and acetonitrile (from 90% to 30% aqueous NaClO4 in 10 min) on a LiChrospher 100 RP-18 cartridge (125 mm x 4.6 mm). Analytes were determined with a UV detector set at 245 nm. Quantitation was accomplished by internal standardization with methionine. Linearity was studied in the range 60-120% of the concentrations expected for gly and thr (viz. for gly from 200 to 400 microgml(-1), and for thr from 100 to 200 microgml(-1)). In reference aqueous samples, linear correlation (r) was better than 0.99 for gly and thr, while in spiked matrix samples r ranged from 0.97 to 0.98. Recoveries were in the 95-105% interval, and precision (CV%, N=6) was better than 5% for both analytes either in cream, ointment or bandages. The method was successfully used for the quality control of topical dermatological preparations.
Assuntos
Fármacos Dermatológicos/análise , Glicina/análise , Preparações Farmacêuticas/análise , Treonina/análise , Acetonitrilas/química , Administração Tópica , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Excipientes/química , Isotiocianatos/química , Cloreto de Metileno/química , Pomadas , Percloratos/química , Controle de Qualidade , Padrões de Referência , Sensibilidade e Especificidade , Compostos de Sódio/química , Espectrofotometria Ultravioleta/métodos , Água/químicaRESUMO
The development and characterization of an anti-aflatoxin B1 (anti-AFB1) immunoaffinity monolithic disk is reported. Polyclonal anti-AFB1 was covalently immobilized in batch on an epoxy-activated monolithic Convective Interaction Media (CIM) disk (12 mm x 3 mm i.d.) by a one-step reaction via epoxy groups of the polymer surface. 0.96 mg of antibody were immobilized and the binding capacity of the CIM disk was determined by frontal analysis. The CIM disk was coupled through a switching valve to a reversed-phase column, namely Chromolith Performance RP-18e. A fully automated HPLC method with fluorescence detection for the determination of aflatoxin B1 in aqueous solution was developed. The total analysis time with the integrated system is 46 min and the retention time of AFB1 is approximately 29 min. The binding capacity of the immunoaffinity disk was evaluated in terms of linearity, precision and accuracy of the extraction procedure. The immunoaffinity support was stable after repeated runs.
Assuntos
Aflatoxina B1/isolamento & purificação , Imunoquímica/instrumentação , Aflatoxina B1/imunologia , Anticorpos/química , Cromatografia Líquida de Alta Pressão , Fluorescência , Corantes Fluorescentes , Indicadores e Reagentes , Soluções , Água , beta-CiclodextrinasRESUMO
Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction liquid chromatography HILIC-UV method was validated in terms of accuracy, precision, linearity, LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their neo-glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for the estimation of the degradation of the proteins under critical conditions (i.e. freeze-thaw cycles) and for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample preparation. In addition, the chromatographic analysis allowed to calculate the yields of the protein glycosylation reaction.
Assuntos
Cromatografia Líquida de Alta Pressão , Glicoproteínas , Interações Hidrofóbicas e Hidrofílicas , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.
Assuntos
Reatores Biológicos , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Tripsina/química , Algoritmos , Sequência de Aminoácidos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hormônio do Crescimento/química , Hormônio do Crescimento/isolamento & purificação , Hormônio do Crescimento/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Mapeamento de Peptídeos/instrumentação , Mapeamento de Peptídeos/métodos , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/metabolismo , Reprodutibilidade dos Testes , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Albumina Sérica/metabolismo , Tripsina/metabolismoRESUMO
S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.
Assuntos
Acetilcisteína/análogos & derivados , Cromatografia Gasosa/métodos , Hipuratos/química , Acetilcisteína/isolamento & purificação , Benzeno/efeitos adversos , Monitoramento Ambiental/métodos , Hipuratos/urina , Espectrometria de Massas/métodos , Exposição Ocupacional/análiseRESUMO
Rat, rabbit and human serum albumins were immobilized on an HPLC stationary phase, and the resulting phases were tested for their abilities to determine the extent and enantioselectivity of ligand binding to the respective albumins. A series of achiral and chiral compounds were chromatographed on the phases including benzodiazepinones, non-steroidal anti-inflammatory drugs, amino acids, warfarin and leucovorin. The chromatographic retentions of the benzodiazepinones and one series of nonsteroidal anti-inflammatory agents were compared with protein binding data from ultrafiltration studies. The observed correlation factors (r) were consistently 0.999, indicating that the albumin phases can be used to determine the magnitude of binding to the respective proteins. The enantioselectivity was also investigated, and the results indicate that the stationary phases can be used to determine relative enantioselectivities and intraspecies differences in this stereoselectivity. For example, when R- and S-warfarin were studied, R-warfarin was retained to a greater extent than S-warfarin by the rabbit serum albumin-stationary phase, whereas the opposite enantioselectivity was found for the rat and human albumins. Binding interaction studies were also conducted on the rabbit and rat albumin stationary phases by sequentially adding increasing concentrations of octanoic acid to the chromatographic mobile phase. The octanoic acid reduced the retention of a series of non-steroidal anti-inflammatory agents, and the results of the experiments suggest that the interaction takes place at two or more sites on the albumin molecule and by anti-cooperative allosteric interactions and competitive displacement. The results of this study demonstrate that the immobilized serum albumin columns can be used to quantitate and probe ligand binding interactions.
Assuntos
Ligantes , Albumina Sérica/química , Animais , Anti-Inflamatórios não Esteroides/química , Benzodiazepinonas/química , Ligação Competitiva , Caprilatos/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Ligação Proteica/efeitos dos fármacos , Coelhos , Ratos , Estereoisomerismo , Ultrafiltração , Varfarina/químicaRESUMO
In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.
Assuntos
Enzimas Imobilizadas/química , Compostos de Epóxi/química , Penicilina Amidase/química , Dióxido de Silício/química , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Microesferas , Tamanho da Partícula , Espectrofotometria Ultravioleta , Estereoisomerismo , Propriedades de SuperfícieRESUMO
In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Enzimas Imobilizadas/metabolismo , Penicilina Amidase/metabolismo , Catálise , Concentração de Íons de Hidrogênio , Hidrólise , Estereoisomerismo , Especificidade por SubstratoRESUMO
The chiral recognition properties of a new chiral stationary phase based on immobilized penicillin G acylase were investigated using 35 acidic racemates. Twenty-seven compounds were resolved with high separation factors. The influences of mobile phase pH, type of organic modifier and ionic strength on enantioselective retention were studied. The most important tool for affecting the enantioselectivity was the mobile phase pH and interestingly the retention order of the enantiomers of some analytes could be controlled by this parameter. The analysis time for resolving enantiomers could be adjusted with a minor decrease in enantioselectivity using a high ionic strength mobile phase buffer while both retention and enantioselectivity decreased by adding organic modifier to the mobile phase. Displacement studies have demonstrated that the enzymatically active site and the chiral adsorption site overlap.
Assuntos
Penicilina Amidase/química , Propionatos/química , Cromatografia Líquida/métodos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.
Assuntos
Reatores Biológicos , Proteínas/química , Tripsina/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Fatty acid-binding proteins (FABPs) are members of a super family of lipid-binding proteins, and occur intracellularly in vertebrates and invertebrates. This review briefly addresses the structural and molecular properties of the fatty acid binding proteins, together with their potential physiological role. Special attention is paid to the methods used to study the binding characteristics of FABPs. An overview of the conventional (Lipidex, the ADIFAB and ITC) and innovative separation-based techniques (chromatographic and electrophoretic methods) for the study of ligand-protein interactions is presented along with a discussion of their strengths, weak points and potential applications. The best conventional approaches with natural fatty acids have generally revealed only limited information about the interactions of fatty acid proteins. In contrast, high-performance affinity chromatography (HPAC) studies of several proteins provide full information on the binding characteristics. The review uses, as an example, the application of immobilized liver basic FABP as a probe for the study of ligand-protein binding by high-performance affinity chromatography. The FABP from chicken liver has been immobilized on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to FABP. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure-retention relationship (QSRRs) correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results. The results of these studies may shed light on the proposed roles of these proteins in biological systems and may find applications in medicine and medicinal chemistry. This knowledge will yield a deeper insight into the mechanism of fatty acid binding in order to indisputably show the central role played by FABPs in cellular FA transport and utilization for a proper lipid metabolism.
Assuntos
Proteínas de Transporte/metabolismo , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese/métodos , Proteínas de Ligação a Ácido Graxo , Ligantes , Modelos Moleculares , Ligação ProteicaRESUMO
A review of Penicillin G Acylase (PGA)-based stationary phases is given, focusing on immobilisation methods, selection of immobilisation material and applications in chiral liquid chromatography. Two immobilization methods, namely "in situ" and "in batch" techniques, are described for the immobilisation of PGA on silica supports. Microparticulate and monolithic silica, both functionalized with aminopropyl- and epoxy-groups, were used in the development of the PGA immobilised enzyme reactor (IMER). The best results, in terms of PGA immobilised amount and enzyme activity, were obtained with the "in situ" immobilisation on epoxy monolithic silica. The use of PGA columns as enzyme reactors for the preparation of 6-APA and for the production of enantiomeric pure drugs in a one-step reaction in described. The review also covers the application of PGA-columns as chiral stationary phases for the separation of acidic enantiomers. An on-line chromatographic system based on the PGA-IMER combined with a switching valve to an analytical column is also described as a highly efficient tool to study the enantioselective hydrolyses properties of PGA. Finally a molecular modelling study is reported with the aim to give more insights into PGA-substrates interactions and to expand the application of these stationary phases as a chiral biocatalysts for pharmaceutical processes.
Assuntos
Enzimas Imobilizadas/análise , Enzimas Imobilizadas/química , Penicilina Amidase/análise , Penicilina Amidase/química , Cromatografia Líquida/métodosRESUMO
A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.
Assuntos
Antituberculosos/análise , Cromatografia Líquida de Alta Pressão/métodos , Isoniazida/análise , Pirazinamida/análise , Rifampina/análise , Combinação de Medicamentos , Preparações Farmacêuticas/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Three chiral calcium antagonist drugs, gallopamil and two dihydropyridine derivatives, have been successfully separated within short retention times using both the alpha 1-acid glycoprotein chiral stationary phase (Chiral-AGP) and the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component, in order to modulate the retention properties of each system. Optimization of pH and organic modifier is carried out using the modified simplex method, with Kaiser's peak separation function as a criterion. The influence of pH and percentage of organic modifier on retention, selectivity, resolution and column performance are discussed for the two dihydropyridines analysed on Chiral-AGP and Ultron ES-OVM stationary phases. A new method is proposed as a new chiral system suitability test for these protein-based phases, utilizing a racemic mixture of closely eluting verapamil enantiomers as a probe.
Assuntos
Bloqueadores dos Canais de Cálcio/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Orosomucoide/química , Ovomucina/química , Estereoisomerismo , Bloqueadores dos Canais de Cálcio/análise , Di-Hidropiridinas/análise , Di-Hidropiridinas/isolamento & purificação , Galopamil/análise , Galopamil/isolamento & purificação , Concentração de Íons de Hidrogênio , Verapamil/análise , Verapamil/isolamento & purificaçãoRESUMO
The development of a new chromatographic reactor based on immobilized Candida rugosa lipase (CRL) is described. The chromatographic system has been used to evaluate the rate differences by which the product enantiomers of esterolytic reactions catalyzed by immobilized CRL are obtained. The method has been applied to a series of racemic 2-aryloxyalkanoic acids and isosteric analogous methyl esters and to some non-steroidal antiinflammatory drugs 2-arylpropanoic acids methyl esters in order to study the structure effects on reaction rate and enantioselectivity. Lipase from C. rugosa has been non-covalently and covalently immobilized on HPLC chromatographic silica supports to develop an immobilized enzyme reactor (IMER). The reactor was connected through a switching valve to an analytical reversed-phase column, which was used for the on-line determination of the hydrolysis rate by peak area integration. The enantiomeric excess of the hydrolytic reaction products was determined off-line on a CSP utilizing immobilized penicillin G acylase (PGA-CSP).