Zi-qing Wang,Li-ping Hao,Zi-xuan Meng,Hao-ran Zhang,Wei-jun Kang,Lian-feng Ai
An LC-MS/MS method was developed and validated for simultaneously quantifying 25-hydroxyvitamin D2, D3, and 1α,25-dihydroxyvitamin D3 in human serum. Protein in 200 μL serum was precipitated with acetonitrile. After centrifugation, the metabolites were derivatized using (diacetoxyiodo)benzene (PIA) and 4-(4-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMAT) and then quantified by the LC-MS/MS system. The limits of detection (LODs) for the three substances were 10, 10, and 5 pg/mL, and the limits of quantification (LOQs) were 20, 20, and 10 pg/mL. The standard curves for these compounds showed linear regression coefficients (R2)>0.998 over specific concentration ranges. Recoveries were 94.36 %–102.34 % for 25(OH)D2, 92.43 %–103.41 % for 25(OH)D3, and 88.98 %–94.36 % for 1α,25(OH)2D3. The mean serum levels in 109 subjects (consisting of 61 healthy adult males and 59 healthy adult females) were 2.0 ± 1.5 ng/ml (25(OH)D2), 16.4 ± 6.1 ng/ml (25(OH)D3) and 36.6 ± 15.1 pg/ml (1α, 25(OH)2D3).Derivatization of vitamin D metabolites using PIA and DMAT is useful for rapidly determining the serum 25(OH) D2, 25(OH) D3 and 1α,25(OH)2D3 concentrations simultaneously in human serum.
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
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