A validated HPLC method for simultaneous estimation of Melatonin and Octyl Methoxycinnamate in combined pharmaceutical applications

A simple, fast and precise reverse phase high performance liquid chromatographic method has been developed for the simultaneous determination of Melatonin and Octyl Methoxycinnamate. Melatonin has become an attractive substrate in sunscreen formulations because of its high antioxidant and photo-protection properties. Octyl methoxycinnamate is one of the chemical UV filter that can be found most of the sunscreen formulations up to 7.5 % according Food and Drug Administration. The aim of the present study was to develop and validate a High-Performance Liquid Chromatography method for the determination of Melatonin and Octyl Methoxycinnamate in combined pharmaceutical or cosmetic applications. As a model of combined pharmaceutical applications, a microemulsion consisting of Melatonin and Octyl Methoxycinnamate was also prepared and characterized in terms of droplet size, pH and viscosity. The separation was performed with a Waters XTerra RP C18 (5 μm, 4.6 x 150 mm). All HPLC assays were performed with 10 μl injection volume, mobile phase consisting of acetonitrile and water, using gradient elution starting at 20% and ending at 90% of acetonitrile with a flow rate of 1.5 ml min-1.The eluent was monitored with UV detection at 222 nm for Melatonin and 306 nm for Octyl Methoxycinnamate. The method was validated according to ICH guidelines. Validation parameters were specificity, accuracy, precision (repeatability and reproducibility), linearity, limit of detection (LOD) and limit of quantification (LOQ). Analytical method development results indicated that the LOD values were 0.132 and 0.049 μg/ml; LOQ values were 0.4 and 0.15 μg/ml and assay exhibited a linear range of 0.5- 60 μg/ml for Melatonin and Octyl Methoxycinnamate, respectively.