Modernizing USP Melatonin Monograph Assay and Impurities Methods (II) – Analytical performance and Analysis of Dietary Supplements

Significance of the topic

Melatonin is widely used as a dietary supplement to regulate sleep cycles and circadian rhythms. Accurate and efficient quantification is essential for product safety, compliance with compendial standards, and consumer protection.

Objectives and overview of the study

This application note presents a modernization of the USP Melatonin monograph assay and impurities methods using optimized HPLC conditions. The aim was to investigate analytical performance and apply the assay to six commercial supplements spanning liquid, tablet, softgel, and capsule formats with melatonin levels from 1 to 10 mg per serving.

Methodology and instrumentation

Analysis was carried out on a Waters Arc HPLC system combined with a 2998 photodiode array detector and an XBridge BEH C18 column (2.5 μm, 4.6 mm × 75 mm). Isocratic separation employed a 22/78 v/v mixture of acetonitrile and 0.5 g/L potassium phosphate buffer at pH 3.5. Sample preparations involved methanolic extraction, vortex mixing, centrifugation, and single point calibration in line with USP recommendations. Empower 3 CDS software was used for data acquisition, spectral library matching, and peak purity assessment.

Key results and discussion

Specificity was confirmed via retention time consistency and PDA spectral matching with no coelution observed. Linearity over 0.001 to 0.1 mg/mL yielded R² > 0.999 with relative errors within ±5%. The limit of quantification was determined at 0.5 μg/mL according to FDA Q2(R1) guidelines. Accuracy studies via spiking demonstrated recoveries between 98.2 and 106.6% with RSD below 5.8%. Six commercial supplements showed melatonin content exceeding label claims by 3 to 42%, with repeatability RSDs below 4%.

Benefits and practical applications

The modernized assay enables rapid, cost-effective, and reliable quantification of melatonin in diverse supplement matrices. Its compliance with USP performance criteria makes it suitable for routine quality control in pharmaceutical, nutraceutical, and regulatory testing laboratories.

Future trends and applications

Future developments may include integration with mass spectrometric detection for comprehensive impurity profiling, adoption of high-throughput workflows, further reduction of solvent consumption, and alignment with evolving regulatory requirements for dietary supplements.

Conclusion

This study confirms that the modernized USP Melatonin assay meets compendial performance requirements, offering a robust and efficient method for the analysis of melatonin in dietary supplements.

Reference

• Stobbe M Melatonin poisoning reports are up in kids study says Associated Press June 2 2022
• LaMotte S Potentially dangerous dose of melatonin and CBD found in gummies sold for sleep CNN April 25 2023
• Melatonin Monograph United States Pharmacopeia Docid GUID454646BEF1DF458C90111FBBCCEFE5BC4 en US 2023
• Yang J Rainville P Modernizing USP Melatonin Monograph Assay and Impurities Methods for Increased Throughput and Reduced Solvent Waste Waters Corporation Application Note 720007978 2023
• US FDA Q2(R1) Validation of Analytical Procedures Text and Methodology Guidance for Industry Sept 2021
• Cerezo AB Leal A Alvarez-Fernandez MA Hornedo-Ortega R Troncoso AM Garcia-Parrilla MC Quality control and determination of melatonin in food supplements J Food Compost Anal 2016 45 80–86
• Erland LA Saxena PK Melatonin natural health products and supplements presence of serotonin and significant variability of melatonin content J Clin Sleep Med 2017 13(2) 275–281
• Cohen PA Avula B Wang YH Katragunta K Khan I Quantity of Melatonin and CBD in Melatonin Gummies Sold in the US JAMA 2023 329(16) 1401–1402
• Andrews KW Gusev PA McNeal M Savarala S Dang PTV Oh L Atkinson R Pehrsson PR Dwyer JT Saldanha LG Betz JM Costello RB Douglass LW Dietary Supplement Ingredient Database DSID and the Application of Analytically Based Estimates of Ingredient Amount to Intake Calculations J Nutr 2018 148(2) 1413S–1421S