The development of simple HPLC methods to separate methylene blue and its metabolites

Abstract

The possible treatment and prevention of Alzheimer’s disease (AD) is one of many clinical applications of methylene blue (MB) that has recently attracted much interest. Due to its ability to interact with various targets, MB exhibits multiple mechanisms by which the progression of neurodegenerative diseases may be decreased. Recently, it has been reported that azure B (AB), the major metabolite of MB, possesses superior effects at various pharmacological targets compared to MB. This finding raises the question that much of the documented pharmacological effects of MB observed in previous studies may in fact be due to the actions of AB. The structural similarities between MB and its metabolites have made the analysis of these compounds very challenging. The published analytical methods for MB and its metabolites have significant disadvantages such as low sensitivity, uneconomically high costs, the need of professionally trained personnel and very expensive, high technology apparatus. These disadvantages delays and limits the research into MB as a drug for the treatment of AD and other neurodegenerative disorders, and also hampers investigations into the pharmacology of MB. In this study, simple and cost effective analytical methods were developed to analyse and separate MB and its metabolites. An accurate, sensitive and reliable high performance liquid chromatography (HPLC) method with which MB and its metabolites were successfully separated, was developed and fully validated. A Synergi Polar-RP column (150 x 4.6 mm, 4 μ, 80 Å) and a mobile phase composed of two parts: ammoniumacetate that is dissolved in a mixture of water and methanol (part A) and a mixture of acetonitrile and methanol (part B). The analysis were done on a Hitachi Chromaster chromatographic system. Also, successful normal phase and reverse phase thin layer chromatography (TLC) methods were developed as a crude method for accessing the purity of MB.