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

Multi-method strategy enhances accuracy in human milk vitamin research. • Mass spectrometry achieved precise 25(OH)D₂/₃ quantification in human milk. • Immunoassays offered rapid screening but showed a 72.98% positive bias. • Liquid chromatography profiled vitamins A and E but lacked sensitivity for D and K. • 25(OH)D₃ increased slightly, while 25(OH)D₂ declined across lactational stages. 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)D 2 and 25(OH)D 3 in human milk, providing critical data to assess infant nutritional intake and guide clinical interventions. Download: Download high-res image (160KB) Download: Download full-size image