Liquid chromatography (ion mobility) coupled to organic and inorganic mass spectrometry for determination of eight thyroid hormones in human milk with enantiomeric separation of thyroxine

Abstract

Human milk is a crucial source of thyroid hormones, essential for neonatal development. We developed and validated a three-phase hollow-fiber liquid-phase microextraction method, coupled to ultra-high performance liquid chromatography and ion mobility mass spectrometry, to simultaneously quantify eight thyroid hormones, including the chiral forms D-thyroxine and L-thyroxine in human milk. The method showed excellent linearity (R2 > 0.999), low limits of detection (0.7–19.8 μg L-1), and high recoveries (83–114 %). The analysis of 30 human milk samples revealed that D-thyroxine (115.1–157.5 μg L-1) was more abundant than L-thyroxine (47.9–193.6 μg L-1), and reversed triiodothyronine prevailed over triiodothyronine. This is the first report of chiral forms of thyroxine, diiodotirosine, and thyronine in human milk, with potential implications for lipid metabolism and infant thyroid regulation. These findings highlight the method sensitivity and biological relevance, offering a robust tool for future nutritional and endocrine studies.