The Development of a Simple High Performance Liquid Chromatography Method to Separate Methylene Blue and Its Metabolites

Abstract

Abstract A simple, rapid and cost-effective high-performance liquid chromatography (HPLC) method was developed and fully validated for the analysis of methylene blue (MB) and its metabolites. The compounds were separated on a Phenomenex Synergi polar-RP (150 mm x 4.6 mm, 4 µm) column, operating at 25ºC. The mobile phase (MP) was a mixture that consisted of 60% part A: 5 mM ammonium acetate dissolved in a water-methanol solution, and 40% part B: acetonitrile-methanol (15:25 v/v), at a flow rate of 0.65 ml/min. The method was validated and the specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, recovery, robustness and stability were determined. The LOD for all the compounds were 0.1 µg/ml and a linear calibration curve over the range 10-500 µg/mL was obtained with a corelation coefficient (R2) > 0.998. The relative standard deviations (RSDs) based on five replicate determinations for each compound across the working range, were less than 2%. Acceptable intra-day and inter-day precision and accuracy were obtained. Variable recoveries between 90-125% across the 50-500 µg/ml range were obtained due to the impurity profiles. The validated method has successfully been utilized for the analysis and separation of methylene blue and its metabolites.