RESUMEN
The 1H NMR spectra obtained from 56 different paracetamol tablets were thoroughly investigated to analyse and quantify besides paracetamol, the excipients and eventual minor components present in the formulations. In the NMR spectra the amide-iminol tautomerism of paracetamol was observed, with the iminol form present at a quantity of only 0.80% of the amide form. Furthermore, seventeen different components of the tablets were identified, ranging from major excipients like starch and polyvinylpyrrolidone (PVP) to minor components like different parabens, sorbate and triacetin. The similarity from all spectra in relation to each of the other spectra was calculated and based on the similarity table a hierarchical cluster analysis (HCA) was performed. Each of the components of the DMSO extracts of the tablets was quantified, providing a component table, which also was used for HCA. The quantitative analysis of paracetamol was determined to have a precision of 0.2% using the residual solvent signals as internal standard. The HCA of the similarity data and the component table were compared with the HCAs obtained by analysis of the crude NMR data. This clearly shows the limitations of multivariate data analysis and the strength of similarity calculations combined with differential NMR, especially in relation to the analysis of trace components. In fact, the spectrum is not a series of unrelated variables, but a superposition of a limited number of component spectra, and the quantities of these components were determined.
Asunto(s)
Acetaminofén , Excipientes , Acetaminofén/química , Análisis por Conglomerados , Excipientes/química , Espectroscopía de Resonancia Magnética , Comprimidos/químicaRESUMEN
Adulteration of essential oils is a common and serious problem. Adequate and fast methods are required to establish the authenticity and purity. GC-MS, 1H and 13C NMR were compared in combination with similarity calculations as well as differential spectroscopy and chromatography for the authentication and determination of purity of vetiver essential oils. In the investigation of eight commercial oils 1H and 13C NMR adequately detected all six adulterants encountered in four of the commercial samples, while GC-MS was not able to detect adulteration with vegetable oils. A great advantage of the combined use of similarity calculation and NMR is that the authenticity can be verified without the need of concomitant measurement of a standard sample. The calculation can be carried out with a registered reference spectrum, in the case of 1H NMR acquired using the same magnetic field, but in the case of 13C NMR the spectrum can be obtained with another magnetic field. 1H NMR has the advantage of high speed, and the results can be obtained within minutes. 13C NMR was found to be superior in its ability to provide unequivocal identification of eventual adulterants, with differential spectroscopy revealing all signals of the adulterants.
Asunto(s)
Aceites Volátiles , Contaminación de Medicamentos , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Aceites de PlantasRESUMEN
The illegal trade in counterfeit and fake drugs is a worldwide multi-billion dollar industry, not only generating enormous economic losses, but health problems for the general population, through direct toxicity, treatment failure and the increased generation of antibiotic resistance. Techniques for high-throughput testing of suspect medicines are needed to face the challenges of the problem. In this study we show that with nuclear magnetic resonance spectroscopy (NMR) drug compliance can be verified in a few minutes, providing data on drug identity, purity and quality without the necessity to develop a specific methodology and using a direct extraction with deuterated solvent. The evaluation of the data is facilitated by similarity calculations and differential NMR spectroscopy. The viability and limitations of this method were assessed, with the application on five different drugs, namely sertraline hydrochloride, alprazolam, vitamin D3, enalapril maleate and paracetamol, in which the individual dosage quantity of the active ingredient ranged from 750 mg down to 0.25 mg. The appropriate sample weight, solvent and internal standard were determined for each drug and quantification was carried out by choosing the most adequate NMR signals for each compound and the internal standard. With the method the accuracy of the quantification is somewhat sacrificed for increased speed in the analysis, but the measurements offer excellent precision and offer the possibility of external calibration. Spectral similarity calculations and differential NMR spectroscopy were used to compare different generic brands and detect eventual undeclared constituents and contaminants. In one brand of alprazolam tablets the undeclared constituent tristearin was found, while in paracetamol tablets the contaminant para-aminophenol was found at a level above the allowed by the legislation. The applicability and limitations of the method are discussed.
Asunto(s)
Medicamentos Falsificados , Humanos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Espectroscopía de Protones por Resonancia Magnética , ComprimidosRESUMEN
Metabolomics is the area of research, which strives to obtain complete metabolic fingerprints, to detect differences between them, and to provide hypothesis to explain those differences [1]. But obtaining complete metabolic fingerprints is not an easy task. Metabolite extraction is a key step during this process, and much research has been devoted to finding the best solvent mixture to extract as much metabolites as possible.Here a procedure is described for analysis of both polar and apolar metabolites using a two-phase extraction system. D2O and CDCl3 are the solvents of choice, and their major advantage is that, for the identification of the compounds, standard databases can be used because D2O and CDCl3 are the solvents most commonly used for pure compound NMR spectra. The procedure enables the absolute quantification of components via the addition of suitable internal standards. The extracts are also suitable for further analysis with other systems like LC-MS or GC-MS.
Asunto(s)
Biomarcadores/análisis , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Extractos Vegetales/metabolismo , Plantas/metabolismo , Solventes/químicaRESUMEN
Cultures from the cyanobacterial strain Microcystis aeruginosa PCC 7806 submitted to nutrient limitation become chlorotic. When returned to nutrient rich conditions these cultures regain their green colour. The aim of this study was to verify whether the cells in these cultures could be considered resting stages allowing the survival of periods of nutrient starvation as has been reported for Synechococcus PCC 7942. The experiments with Microcystis were carried out in parallel with Synechococcus cultures to rule out the possibility that any results obtained with Microcystis were due to our particular experimental conditions. The results of the experiments with Synechococcus PCC 7942 cultures were comparable to the reported in the literature. For Microcystis PCC 7806 a different response was observed. Analysis of chlorotic Microcystis cultures by flow cytometry showed that the phenotype of the cells in the population was not homogenous: the amount of nucleic acids was about the same in all cells but only around one percent of the population emitted red autofluorescence indicating the presence of chlorophyll. Monitoring of the reversion of chlorosis by flow cytometry showed that the re-greening was most likely the result of the division of the small population of red autofluorescent cells originally present in the chlorotic cultures. This assumption was confirmed by analysing the integrity of the DNA and the membrane permeability of the cells of chlorotic cultures. Most of the DNA of these cultures was degraded and only the autofluorescent population of the chlorotic cultures showed membrane integrity. Thus, contrary to what has been reported for other cyanobacterial genera, most of the cells in chlorotic Microcystis cultures are not resting stages but dead. It is interesting to note that the red autofluorescent cells of green and chlorotic cultures obtained in double strength ASM-1 medium differ with respect to metabolism: levels of emission of red autofluorescence are higher in the cells of green cultures and the ability to convert fluorescein diacetate of these cells are heterogeneous when compared to the autofluorescent cells of chlorotic cultures. Thus, the small population of the red autofluorescent cells of chlorotic cultures are in a differentiated metabolic state that allow them to persist in conditions in which most of the population loses viability; persistent cells can be detected in chlorotic cultures maintained for more than a year.
Asunto(s)
Microcystis/fisiología , Synechococcus/metabolismo , Clorofila/química , Clorofila A , Color , ADN/metabolismo , Citometría de Flujo , Glucosa/química , Espectroscopía de Resonancia Magnética , Microcystis/metabolismo , Microscopía Fluorescente , Fenotipo , Polímeros/químicaRESUMEN
From the aerial parts of Pentas lanceolata, belonging to the family Rubiaceae, a series of iridoid glucosides was isolated by preparative HPLC. Seven iridoid glucosides were identified. Besides asperuloside and asperulosidic acid, characteristic iridoids for Rubiaceae, five new iridoids were isolated, namely, tudoside (1), 13R-epi-gaertneroside (2), 13R-epi-epoxygaertneroside (3), and a mixture of E-uenfoside (4) and Z-uenfoside (5). Further, it was shown that the compound reported as citrifolinin B (6) is in fact the same as tudoside and should be revised. Also, the configuration of the previously reported iridoids gaertneroside and epoxygaertneroside has been elucidated.
Asunto(s)
Iridoides/aislamiento & purificación , Rubiaceae/química , Brasil , Iridoides/química , Estructura Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
[Structure: see text] Citrifolin A was revised to dehydromethoxygaertneroside (2). Citrifolinoside and yopaaoside A were found to be identical, and their structures were revised to the new structure dehydroepoxymethoxygaertneroside (5). Yopaaoside B was revised to citrifolinoside A (9), and morindacin was revised to borreriagenin (11).
Asunto(s)
Glucósidos/química , Iridoides/química , Monoterpenos/química , Morinda/química , Piranos/química , Glucósidos/metabolismo , Glucósidos Iridoides , Iridoides/metabolismo , Estructura Molecular , Monoterpenos/metabolismo , Morinda/metabolismo , Piranos/metabolismoRESUMEN
The frequency of cyanobacterial blooms has been increasing all over the world. These blooms are often toxic and have become a serious health problem. The aim of this work was to search for population density control mechanisms that could inhibit the proliferation of the toxic bloom-forming genus Microcystis. Microcystis PCC 7806 cultured for long periods in liquid ASM-1 medium loses its characteristic green colour. When a medium of chlorotic cultures is added to a nutrient-replete culture, cell density increase is drastically reduced when compared with controls. Inhibition of cell proliferation occurs in Microcystis cultures from any growth stage and was not strain-specific, but other genera tested showed no response. Investigations on the mechanism of growth inhibition showed that cultures treated with the conditioned medium acquired a pale colour, with pigment concentration similar to that found in chlorotic cultures. Ultrastructural examination showed that the conditioned medium induced thylakoid membrane disorganization, typical of chlorotic cells, in nutrient-replete cultures. An active extract was obtained and investigations showed that activity was retained after heating and after addition of an apolar solvent. This indicates that activity of the conditioned medium from chlorotic cells results from non-protein, apolar compound(s).
Asunto(s)
Medios de Cultivo Condicionados/toxicidad , Eutrofización/fisiología , Microcystis/crecimiento & desarrollo , Pigmentación/efectos de los fármacos , Medios de Cultivo Condicionados/química , Microcystis/efectos de los fármacos , Microcystis/ultraestructura , Microscopía Electrónica , Dinámica Poblacional , Especificidad de la Especie , Tilacoides/efectos de los fármacosRESUMEN
A complete procedure is described for the extraction, detection and quantification of anatoxin-a in biological samples. Anatoxin-a is extracted from biomass by a routine acid base extraction. The extract is analysed by GC-MS, without the need of derivatization, with a detection limit of 0.5 ng. A method was developed for the accurate quantification of anatoxin-a in the standard solution to be used for the calibration of the GC analysis. 1H NMR allowed the accurate quantification of microgram quantities of anatoxin-a. The accurate quantification of compounds in standard solutions is rarely discussed, but for compounds like anatoxin-a (toxins with prices in the range of a million dollar a gram), of which generally only milligram quantities or less are available, this factor in the quantitative analysis is certainly not trivial. The method that was developed can easily be adapted for the accurate quantification of other toxins in very dilute solutions.