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.
The study aimed to evaluate the effects of occupational exposure on the cellular immune response by measuring neopterin release and the kynurenine pathway and to assess trace element status and heavy metal exposure. The study group consisted of 30 male e-waste recycling workers who underwent detailed occupational anamnesis and a comprehensive physical examination. Concentrations of essential trace elements (Cr, Co, Cu, Mn, Mo, Ni, Se, and Zn) and heavy metals (Sb, Cd, and Pb) were measured in whole blood or serum samples using inductively coupled plasma mass spectrometry (ICP-MS). Serum tryptophan (Trp) and kynurenine (Kyn) levels were determined by high-performance liquid chromatography (HPLC), and serum neopterin concentrations were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Serum neopterin, Trp, and Kyn concentrations were 7.8 ± 0.7 nmol/L, 54 ± 2 μmol/L, and 0.5 ± 0.04 μmol/L, respectively. The estimated indoleamine 2,3-dioxygenase (IDO) activity, expressed as the Kyn/Trp ratio, was 8.8 ± 0.8 μmol/mmol. Elevated levels of individual metals, except for Pb, did not significantly alter neopterin release or kynurenine pathway activity (all, p > 0.05). Blood Pb levels showed positive correlations with both Sb and neopterin concentrations, whereas they were negatively correlated with Cu levels (all, p < 0.05). These results indicate that e-waste recycling workers are exposed to multiple heavy metals, suggesting potential inhalation exposure, and that such exposure may influence trace element status, neopterin release, and the kynurenine pathway without inducing pronounced changes. Further studies with larger populations are warranted to confirm these findings and to better characterize the health impact of multiple metal exposure. Occupational safety measures should be strengthened to minimize such exposures.
