Modernizing USP Melatonin Liquid Chromatographic Analyses for Increased Throughput and Reduced Environmental Impact.

Background: Melatonin supplements are often used to alleviate jetlag and other sleep-depletion related disorders. Recent studies found large inconsistencies between labeled values and actual contents of melatonin within products, which has led to concerns over the quality of melatonin supplements. In order to facilitate the quality control testing of melatonin supplements, an improved and more modern approach to the liquid chromatographic analysis of melatonin is required. In addition, growing public concern over the environmental footprint of analytical laboratories exacerbates the need to modernize legacy analytical procedures with more eco-friendly or greener approaches.

Objective: This study aims to optimize the routine liquid chromatographic analyses that are prescribed in the US Pharmacopeia (USP) melatonin monograph on a High Performance Liquid Chromatography (HPLC) system without fundamentally modifying the methods.

Method: The melatonin assay and the melatonin related compounds (impurities) test were optimized on a C18 column packed with 2.5 µm particles. The column length and the gradient elution parameters were adjusted following the guidelines on Adjustment of Chromatographic Conditions in USP General Chapter <621>. The mobile phase compositions were optimized to meet the system suitability requirements that were specified in the USP melatonin monograph. The flow rate was optimized for better separation efficiency.

Results: The optimized HPLC methods not only met the USP system suitability requirements in relative retention time (RRT), resolution, and relative standard deviation (RSD), but also demonstrated excellent linearity, sensitivity, accuracy, precision (repeatability), and would have a lower environmental impact.

Conclusions: The optimized HPLC methods for assay of melatonin and test of its related compounds achieved significantly increased throughput and a reduced environmental impact, without fundamentally modifying the methods.

Highlights: The optimized HPLC methods are significantly faster and more eco-friendly. These methods can be implemented on HPLC systems without a full re-validation.