Validated HPLC-MS/MS quantification of melatonin in human breast milk from mothers of preterm infants confirms circadian rhythmicity

Melatonin in human breast milk follows a circadian rhythmicity and serves as a crucial chronobiological signal for neonatal development. Preterm infants are particularly vulnerable to melatonin deficiency due to the immature pineal gland function. Accurate analytical methods are essential for characterizing temporal patterns and assessing clinical relevance. We developed and validated a sensitive LC-MS/MS method for melatonin quantification in breast milk using liquid-liquid extraction with ethyl acetate. The method employed melatonin-d4 as internal standard and scheduled multiple reaction monitoring. Validation followed Eurachem and FDA guidelines and the method was applied to paired daytime and nighttime breast milk samples from mothers of preterm infants ( n  = 80). The method demonstrated excellent analytical performance with an LLOQ of 4.8 pg/ml, intraday and interday precision <5 %, and 97 % extraction efficiency. Nighttime melatonin concentrations (27.5 ± 16.8 pg/ml) were significantly higher than daytime levels (9.0 ± 10.0 pg/ml, p < 0.0001). Circadian variation was maintained across all lactational stages (colostrum, transitional, and mature milk). This validated LC-MS/MS method enables reliable quantification of physiological melatonin concentrations in breast milk and confirms circadian rhythmicity across lactation stages. The simplified sample preparation and robust performance make it suitable for clinical studies investigating chrononutrition and neonatal chronobiological development.