LC-MS/MS outperforms ELISA and UPLC-PDA for targeted quantification of 25(OH)D₂ and 25(OH)D₃ in human milk: A practical analytical strategy.

Authors

Yuanyuan Zhao, Yongmao Lu, Tian Yang, Shaohua Hu, Yuanyuan Yan, Xingguo Wang, Wei Wei

Published in

Food research international (Ottawa, Ont.). Volume 241. Pages 119787. Oct 01, 2026. Epub Jun 15, 2026.

Abstract

Accurate analysis of vitamin D in human milk is essential for infant nutrition but remains analytically challenging due to low levels and a complex matrix. This study evaluates a practical multi-method strategy using LC-MS/MS, ELISA, and UPLC-PDA for vitamin D analysis in human milk. LC-MS/MS precisely quantified 25(OH)D₂ and 25(OH)D₃ with assay LOQ of 0.045 ng/mL and 0.100 ng/mL, respectively. ELISA showed a 11.58 ng/mL positive bias of vitamin D which limits use for accurate quantification. UPLC-PDA profiled vitamins A and E effectively but lacked sensitivity for vitamin D. Longitudinally, LC-MS/MS results show 25(OH)D₃ and total 25(OH)D concentrations remained relatively stable, with medians ranging from 0.141 to 0.281 ng/mL and 0.263 to 0.315 ng/mL, respectively, across the study period, while 25(OH)D₂ peaked at 8 weeks before declining. This study establishes LC-MS/MS as the preferred analytical strategy for quantifying 25(OH)D₂ and 25(OH)D₃ in human milk, providing critical data to assess infant nutritional intake and guide clinical interventions.

PMID:
42562545
Bibliographic data and abstract were imported from PubMed on 07 Aug 2026.

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