Analytical Measurement of Serum 25-OH-vitamin D₃, 25-OH-vitamin D₂ and Their C3-epimers by LC-MS/MS in Infant and Pediatric Specimens

Objectives: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D₃ (25-OH-D₃), 25-OH-vitamin D₂ (25-OH-D₂), and their C3-epimers.

Methods: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D₃ and C3-epi-25-OH-D₃, 407.2→371.3 for d6-25-OH-D₃, 413.2→331.2 for 25-OH-D₂ and C3-epi-25-OH-D₂ and 419.2→337.1 for, d6-25-OH-D₂. As a proof-of-principle, 25-OH-D₃ and C3-epi-25-OH-D₃ were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer.

Results: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D₂/₃ and <15% for C3-epi-25-OH-D₃. C3-epi-25-OH-D₃ was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D₃ concentrations increased linearly, while C3-epi-25-OH-D₃ concentrations remained constant. At 12 months of age, C3-epi-25-OH-D₃ concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D₃ supplements did not contain appreciable amounts of C3-epi-D₃.

Conclusions: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D₃ metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.