Development of Melatonin Quantification in Human Breastmilk Using Liquid Chromatography
Faculty Mentor Information
Dr. Nancy Johnston, Lewis-Clark State College
Presentation Date
7-16-2026
Abstract
Melatonin is a vital hormone regulating circadian rhythms, immune function, and antioxidant pathways. In human breast milk, nocturnal melatonin concentrations peak significantly to assist nursing infants in establishing stable sleep-wake cycles. While triple-quadrupole tandem mass spectrometry (LC-MS/MS) typically provides the standard for high-sensitivity quantification, achieving reliable picogram-level detection remains a significant technical challenge for laboratories limited to single-quadrupole LC-MS systems. This study introduces an optimized extraction and acquisition framework designed to address this limitation. The method features a rigorous sample preparation protocol to isolate melatonin from complex lipid and protein milk matrices using solid-phase extraction (SPE) cartridges. Chromatographic and single ion mass spectrometry parameters—including injection volumes, column temperature, solvent composition and fragmentor voltage—were systematically optimized to maximize sensitivity. Analytical accuracy was validated using a deuterated melatonin internal standard. Ultimately, this protocol will provide a highly reproducible, accessible, and cost-effective methodology for precise, picogram-level melatonin quantification in challenging biological matrices without requiring high-end tandem MS instrumentation.
Development of Melatonin Quantification in Human Breastmilk Using Liquid Chromatography
Melatonin is a vital hormone regulating circadian rhythms, immune function, and antioxidant pathways. In human breast milk, nocturnal melatonin concentrations peak significantly to assist nursing infants in establishing stable sleep-wake cycles. While triple-quadrupole tandem mass spectrometry (LC-MS/MS) typically provides the standard for high-sensitivity quantification, achieving reliable picogram-level detection remains a significant technical challenge for laboratories limited to single-quadrupole LC-MS systems. This study introduces an optimized extraction and acquisition framework designed to address this limitation. The method features a rigorous sample preparation protocol to isolate melatonin from complex lipid and protein milk matrices using solid-phase extraction (SPE) cartridges. Chromatographic and single ion mass spectrometry parameters—including injection volumes, column temperature, solvent composition and fragmentor voltage—were systematically optimized to maximize sensitivity. Analytical accuracy was validated using a deuterated melatonin internal standard. Ultimately, this protocol will provide a highly reproducible, accessible, and cost-effective methodology for precise, picogram-level melatonin quantification in challenging biological matrices without requiring high-end tandem MS instrumentation.