Validated UHPLC Methods for Melatonin Quantification Reveal Regulatory Violations in EU Online Dietary Supplements Commerce

The global sleep aids market has grown significantly due to rising stress and increased awareness of sleep&rsquo:s importance. Melatonin, available in the EU as a prescription or over-the-counter medicine, depending on the country, is also permitted in dietary supplements with country-specific limits. Recent reports indicate concerning levels of excessive melatonin in EU dietary supplements, necessitating accurate quantification methods. We developed and validated, by applying accuracy profiles, ISO17025-compliant, rapid ultra-high performance liquid chromatography (UHPLC) methodologies coupled with either diode array detection (DAD) or high-resolution accurate mass spectrometry (HRAM MS). The cost-effective UHPLC-DAD method is suitable for medicines and most dietary supplements, except those more complex herbal matrices containing passionflower, hop, hemp, lime tree or lavender or their extracts, where UHPLC-HRAM MS is recommended due to selectivity issues of the DAD methodology. To demonstrate the applicability, we analyzed 50 dietary supplements claiming to contain melatonin&mdash:25 from legal supply chains and 25 from suspicious sources claiming therapeutic melatonin content. Our findings confirmed previous reports of high melatonin content in online products, especially when purchased through rogue internet pharmacies. Moreover, 12% of legal supply chain samples violated current legislation through unauthorized health claims or contained at least triple the melatonin amount permitted in Belgium. This research provides reliable analytical methods for regulatory bodies and confirms the circulation of non-compliant melatonin-containing dietary supplements in the EU market, even in the legal supply chain.