Quantification of 24,25-Dihydroxyvitamin D₃ in Serum Using LC-MS/MS With Derivatization and Lipid-Removal Filtration

Accurate assessment of vitamin D metabolism is crucial not only for the diagnosis and treatment of disorders related to bone health and calcium homeostasis but also for understanding its broader physiological roles in immunity, cellular differentiation, cardiovascular regulation, and endocrine function. Although 25-hydroxyvitamin D₃ (25(OH)D₃) is routinely measured in clinical practice, the low-abundance metabolite 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) provides complementary insight into vitamin D catabolism. Reduced or undetectable 24,25(OH)₂D₃ levels may signal impaired CYP24A1 function or insufficient conversion of 25(OH)D₃ to maintain appropriate intracellular concentrations of biologically active 1,25(OH)₂D₃. However, quantification of 24,25(OH)₂D₃ remains analytically challenging and requires highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. In-house developed LC-MS/MS approaches are being increasingly employed, offering greater specificity and sensitivity over conventional immunoassays, allowing accurate detection of low-abundance vitamin D metabolites. Additionally, when interpreted together with 25(OH)D₃, 24,25(OH)₂D₃ allows the calculation of the vitamin D metabolite ratio (VMR), which offers a more accurate assessment of vitamin D sufficiency and catabolism. The aim of this study was to develop and validate a robust LC-MS/MS method using dynamic multiple reaction monitoring (dMRM) and sample derivatization for quantifying 24,25(OH)₂D₃ in human serum. The method described was evaluated according to EMA guidelines and DEQAS controls. The matrix effect was minimized through lipid-removal filtration. The assay demonstrated excellent linearity (R ² = 0.9982), intra- and inter-assay precision below 14%, and LOQ of 0.64 ng/mL. Recovery from DEQAS samples ranged from 80% to 118%, with a matrix-induced ion enhancement of ∼17%. Additionally, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization enhanced the sensitivity 100-fold. This highly sensitive LC-MS/MS method is suitable for clinical and research laboratories equipped with an electrospray ionization (ESI) source. Precise quantification of 24,25(OH)₂D₃ can complement routine 25(OH)D₃ analysis, support VMR determination, and serve as a reliable biomarker for disorders associated with altered vitamin D metabolism.