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 D-3 (25-OH-D-3), 25-OH-vitamin D-2 (25-OH-D-2), 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-D-3 and C3-epi-25-OH-D-3, 407.2 -> 371.3 for d(6)-25-OH-D-3, 413.2 -> 331.2 for 25-OH-D-2 and C3-epi-25-OH-D-2 and 419.2 -> 337.1 for, d(6)-25-OH-D-2. As a proof-of-principle, 25-OH-D-3 and C3-epi-25-OH-D-3 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-D-2/3 and <15% for C3-epi-25-OH-D-3. C3-epi-25-OH-D-3 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-D-3 concentrations increased linearly, while C3-epi-25-OH-D-3 concentrations remained constant. At 12 months of age, C3-epi-25-OH-D-3 concentration dropped by almost 50% (11.4 nmol/L in infants <= 1 year of age vs. 5.4 nmol/L in infants 1-2 years of age; p<0.001). Liquid vitamin D-3 supplements did not contain appreciable amounts of C3-epi-D-3. Conclusions: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D-3 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. (c) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.