RESUMEN
According to the USP erythromycin determination in finished oral products as well as in some topical formulations is mainly carried out via microbiological assays. However, these assays are known for their long incubation periods, lack of precision and low sensitivity. In literature one HPLC method for the quantification of erythromycin in creams is described, which depends on electrochemical detection, but HPLC electrochemical detection has not emerged as popular choice in routine analysis. Furthermore two other HPLC-UV methods are described for the isolation of erythromycin from gels and creams involving tedious and time consuming extraction steps, the reason why they are not suited to be applied in routine analysis. This paper describes a new HPLC-UV method for the determination of erythromycin in creams, which implies a much easier extraction procedure than that cited in literature to date, based solely on the solubilization of erythromycin followed by freezing the cream matrix. Validation experiments confirmed the precision and accuracy of the method. Good linearity of the assay was found over the investigated concentration range of 70-130% (corresponding to 0.77-1.43 g of erythromycin A in 100 g cream base). The coefficient of correlation resulting from unweighted linear regression was 0.9998, allowing a one-point calibration in routine analysis. By the implementation of an internal standard in the quantification of erythromycin an improved precision could be achieved in routine analysis. This new analytical method yields cleaner extracts and allows a higher throughput, saving costs, solvents and time and can be thus recommended to all laboratories.
Asunto(s)
Antibacterianos/análisis , Fármacos Dermatológicos/análisis , Eritromicina/análisis , Cromatografía Líquida de Alta Presión , Pomadas/análisis , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrofotometría UltravioletaRESUMEN
Today, NIR-spectroscopy is an established analytical technique not only in the identification of raw materials but also in the quantification of active ingredients in tablets. In this work calibration models were set up with tablets of the same active ingredient but of miscellaneous origin and manufacturess. Consequently the tablets had different excipients and appearance. The pharmaceutical preparations used included atenolol 100 mg tablets, enalapril 20 mg tablets and acetylsalicylic acid (ASS) tablets of different dosage units. In order to proof if the calibration models set up are generally feasible the assay declared by the manufacturer was used to calculate the partial least square (PLS) calibration. With respect to enalapril tablets simultaneous analysis by HPLC, according to USP 26 was carried out. It was investigated if such methods allow a determination of active ingredients in tablets within limits of +/- 10% of declaration. It was shown that it is possible to set up calibration models to quantify active ingredients in tablets independent of adjuvants or optical appearance. Additionally it could be shown that NIR-spectroscopy is also applicable to determine the concentration of active ingredients in blister-packed tablets.
Asunto(s)
Comprimidos/normas , Atenolol/administración & dosificación , Atenolol/química , Calibración , Embalaje de Medicamentos , Enalapril/administración & dosificación , Enalapril/química , Control de Calidad , Reproducibilidad de los Resultados , Espectroscopía Infrarroja CortaRESUMEN
Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.
Asunto(s)
Escherichia coli , Activación del Canal Iónico , Canales de Potasio/química , Canales de Potasio/metabolismo , Membrana Celular/química , Membrana Celular/ultraestructura , Dicroismo Circular , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Potasio/metabolismo , Canales de Potasio/genética , Canales de Potasio/aislamiento & purificación , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes , Solubilidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A validated HPLC method for the determination of 11-keto-beta-boswellic acid (KBA) in human plasma was developed. The method involves the solid-phase extraction of KBA from plasma followed by a separation with reversed-phase HPLC. Calibration was based on external standardisation and ranged between 0.1 and 2.0 microg KBA per ml plasma. Linearity was established over the entire calibration range and in each case the coefficient of correlation (r2) was above 0.99. The recovery of KBA from plasma was 85.7%. It was further demonstrated that the method can be applied successfully to monitor the level of KBA in plasma.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Triterpenos/sangre , Calibración , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
Over the last few years, St. John's Wort products have enjoyed a tremendous surge in interest and sales for the therapy of mild to moderate depression. Although the complete spectrum of active substances in this herbal extract has not yet been elucidated, it is certain that hyperforin is an important component. Further, it appears that the hypericins may also contribute to the antidepressive activity. In this study, the content uniformity of eleven St. John's Wort products sold exclusively in pharmacies was determined. The main objective was to determine the batch-to-batch reproducibility of the various products. The hyperforin was analysed according to a previously published HPLC method, while the total hypericin content was determined by an electrochemical method. The results indicate that some, but not all, products show very reproducible batch-to-batch properties. Also, individual products have different hypericin and hyperforin levels, and are therefore not switchable--even when products are manufactured under similar extraction and processing conditions, have the same raw material:extract ratios (on a dry basis) and contain the same amount of extract per unit dosage form.
Asunto(s)
Química Farmacéutica/normas , Hypericum , Perileno/análogos & derivados , Fitoterapia/normas , Extractos Vegetales/química , Antracenos , Antidepresivos/análisis , Compuestos Bicíclicos con Puentes , Humanos , Perileno/análisis , Floroglucinol/análogos & derivados , Reproducibilidad de los Resultados , Terpenos/análisisRESUMEN
OBJECTIVES: To compare the hyperforin and hypericin content of currently available St. John's wort products and to determine their batch-to-batch reproducibility. DESIGN: Representative products were obtained either directly from the manufacturer or purchased from pharmacies in and around Frankfurt, Germany. For five batches from each of the eight manufacturers, 10 individual dosage forms (tablets or capsules) were analyzed for both hyperforin and hypericin content. SETTING: Laboratories of the Institute of Pharmaceutical Chemistry at Johann Wolfgang Goethe University, Frankfurt, Germany. PRODUCTS: Eight German St. John's wort products containing from 250 mg to 612 mg dry extract were studied. Three of these products are capsules, four are film-coated tablets, and one is a sugar-coated tablet. Two of the products (Jarsin 300 and Neuroplant 300) are also available in the United States. METHODS: Hyperforin concentrations were analyzed by high-performance liquid chromatography. Total hypericin concentrations were determined by polarography, an electrochemical method. Concentrations were compared among different batches of the same product and among products from different manufacturers. RESULTS: The products contained widely differing amounts of hypericin and hyperforin, even after correcting for differences in the amount of extract per dose. Some products demonstrated consistent concentrations of hyperforin and hypericin from batch to batch, others exhibited pronounced interbatch variability. CONCLUSION: The St. John's wort preparations studied exhibited large differences in hypericin and hyperforin content and are not interchangeable for the treatment of mild-to-moderate depression. Pharmacists should take this variability into account when counseling patients on the use of St. John's wort products.
Asunto(s)
Antibacterianos/análisis , Hypericum/química , Extractos Vegetales/análisis , Plantas Medicinales , Terpenos/análisis , Compuestos Bicíclicos con Puentes , Cromatografía Líquida de Alta Presión , Formas de Dosificación , Alemania , Floroglucinol/análogos & derivadosRESUMEN
The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.
Asunto(s)
Alcaligenes/metabolismo , Proteínas Bacterianas/metabolismo , Hemoproteínas/metabolismo , Fosfolípidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cromatografía en Capa Delgada , Cristalografía por Rayos X , Cartilla de ADN , Hemoproteínas/química , Hemoproteínas/genética , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Fosfolípidos/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Urinary organic acid analysis is a vital diagnostic tool in the investigation of patients with suspected inborn errors of metabolism. Recent descriptions of diseases producing abnormal chiral metabolites have provided the need for individual enantiomer analysis. In this paper the isolation and simultaneous stereodifferentiation of lactic, glyceric and 2-hydroxyglutaric acids using capillary gas chromatography on heptakis (2,3-di-O-methyl-6-O-tert.butyldimethylsilyl)-beta-cyclodextrin as the chiral stationary phase is described. The diagnostic potential of this method is demonstrated by the analysis of urine samples from two patients with D-2-hydroxyglutaric aciduria and a dog with D-glyceric aciduria. Due to the lack of specific clinical symptoms in these diseases, enantiomeric analysis is vital for diagnosis enabling prompt therapeutic intervention.