Development and Validation of a Novel Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Serum 3-Epi-25-Hydroxyvitamin D 3 , 25-Hydroxyvitamin D 3 , and 25-Hydroxyvitamin D 2

Background: The epimeric form of 25-hydroxyvitamin D3 (3-epi-25-hydroxyvitamin D3, 3-epi-25-OH-D3) can interfere with the accurate measurement of total vitamin D. Therefore, reliable quantification of epimeric metabolites is important for improving measurement accuracy.Here, we developed and validated a novel laboratory-developed test (LDT) based on an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to measure the concentrations of 3-epi-25-OH-D3, 25-OH-D3, and 25-OH-D2 in serum.Methods: Sample pretreatment included derivatization with the PTAD reagent (4-phenyl-1,2,4-triazoline-3,5-dione).The assay was performed using the Shimadzu LC-40D UPLC system (Shimadzu, Japan) and Sciex Triple Quad 5500+ QTRAP Ready MS/MS system (Sciex, USA), with a total runtime of 16 minutes, flow rate of 0.15 mL/min, and serum volume of 100 µL.We evaluated the accuracy, precision, linearity, limit of detection (LOD), lower limit of quantification (LLOQ), carryover, and matrix effects.The results were compared with those obtained using the MSMS Vitamin D kit (PerkinElmer, Finland).Results: The precision and accuracy were all within acceptable limits.The verified linear ranges were 0.30-24.70,0.29-137.00,and 0.29-137.00ng/mL with LLOQs of 0.30, 0.29, and 0.29 ng/mL for 3-epi-25-OH-D3, 25-OH-D3, and 25-OH-D2, respectively.No significant carryover or matrix effects were observed.The method comparability for the MSMS Vitamin D kit was excellent, validating the transition to the new platform.Conclusions: This novel LDT UPLC-MS/MS assay showed acceptable analytical performance for measuring the serum concentrations of 3-epi-25-OH-D3, 25-OH-D3, and 25-OH-D2 and may help improve the accuracy and comparability of total vitamin D measurements, particularly in largescale epidemiological studies.