High-throughput quantitation of trace level melatonin in human milk by on-line enrichment liquid chromatography-tandem mass spectrometry

Abstract

The level of melatonin in human milk might be closely related to infant development and the building up of their circadian rhythms. The large population investigation on this topic would provide insights for the prevention and treatment of diseases related to early development and circadian rhythms. However, it has not been well studied. Trace level endogenous melatonin and difficulties in sample collection are among the challenges limiting the progress. High throughput analytical method with high specificity and sensitivity to determine the endogenous melatonin concentration is highly desired. A newly developed easily operated and high-throughput sensitive on-line enrichment liquid chromatography-tandem mass spectrometry (LC-MS/MS) method would be reported in this paper. Melatonin-d3 (MEL-d3) was used as a surrogate standard for the calibration curve and melatonin-d4 (MEL-d4) was used as an internal standard. Sample preparation was simply performed in 96-well plate by protein precipitation using acetonitrile (ACN). The supernatant was injected directly into the easily configured LC-MS/MS system with an enlarged sample loop and a mixer. Positive mode multiple reaction monitoring (MRM) was adopted for the measurement of melatonin in milk. 100 μL sample was used for analysis and the calibration curve linear range was 1–1000 pg mL−1. In three validation batches, the accuracy was within 11.0% deviation from the relative nominal concentration, whereas the intra- and inter-assay precision was ≤4.1% and ≤6.8% relative standard deviation (RSD), respectively. Although matrix effect was observed in the validation experiments, the stable isotope labeled internal standard (MEL-d4) could correct it and the overall relative matrix effect of MEL-d3/MEL-d4 was close to 100%. The overall spike recovery of the method was 101.7% with 5.1% RSD. Compared to currently reported methods, it could reach 1 pg mL−1 lower limit of quantification (LLOQ) with a smaller sample volume, sample preparation could be easily performed by automated liquid handling system and was more suitable for large population cohort studies on trace level endogenous melatonin determination.