Measurement of serum 3-epi-25-hydroxyvitamin D_3, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ in infant, paediatric and adolescent populations of Korea using ultra-performance liquid chromatography-tandem mass spectrometry

Background We evaluated the performance of ultra-performance liquid chromatography-tandem mass spectrometry to measure serum 3-epi-25-hydroxyvitamin D₃, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ concentrations in 519 infant, paediatric and adolescent serum samples in Korea. Methods We used a Kinetex XB-C18 column and isocratic methanol/water (77.5/22.5, v/v) with 0.025% (v/v) high-performance liquid chromatography solvent additive flowing at 0.25 mL/min, yielding an 11 min/sample run time. A TQD triple quadrupole mass spectrometer in electrospray ionization positive ion mode with multiple reaction monitoring transition via an MSMS vitamin D kit was used to evaluate precision, carryover, ion suppression and linearity. Samples were prepared using the 4-phenyl-1,2,4-triazoline-3,5-dione derivatization method. Results Intra- and inter-run precisions were 1.23-13.28% and 1.02-10.08%, respectively. Group carryovers were -0.27% and 0.10%, respectively. There was no ion suppression. The calibration curve showed good linearity from calibrator Level 1 (11.75 nmol/L) to 6 (375 nmol/L) with R² > 0.9999. The 3-epi-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃ peaks were clearly separated in the extracted ion chromatogram. Infant serum samples 3-epi-25-hydroxyvitamin D₃ concentrations were significantly higher than paediatric and adolescent concentrations. Conclusions The ultra-performance liquid chromatography-tandem mass spectrometry assay performed acceptably, clearly separating 3-epi-25-hydroxyvitamin D₃ from 25-hydroxyvitamin D₃. High 3-epi-25-hydroxyvitamin D₃ concentrations were observed in infant but not in paediatric and adolescent serum samples.