ABSTRACT
Retigabine was the first neuronal potassium channel opener for the treatment of epilepsy. During the manufacture of retigabine, two unknown impurities were present in laboratory batches in the range of 0.05-0.1% based upon HPLC analysis. These unknown impurities were obtained from the enriched reaction mother liquor by column chromatography. The structure of these process-related impurities were elucidated using FT-IR, (1)H NMR, (13)C NMR, 2D NMR (HSQC, HMBC, NOESY) and MS spectral data. Based on the complete spectral analysis and knowledge of the synthetic route of retigabine, these two new impurities were designated as ethyl 4-fluorobenzyl(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)carbamate (impurity-II) and diethyl 5-((ethoxycarbonyl)(4-fluorobenzyl)amino)-2-oxo-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate (impurity-III). Impurity identification, structure elucidation and the formation of impurities were also discussed.
Subject(s)
Anticonvulsants/standards , Carbamates/isolation & purification , Carbamates/standards , Carboxylic Acids/isolation & purification , Drug Contamination , Imidazoles/isolation & purification , Phenylenediamines/standards , Technology, Pharmaceutical/standards , Anticonvulsants/analysis , Carbamates/analysis , Carbamates/chemistry , Carboxylic Acids/chemistry , Chromatography, High Pressure Liquid , Imidazoles/chemistry , Molecular Conformation , Phenylenediamines/analysis , Spectrometry, Mass, Electrospray IonizationABSTRACT
To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.
