Simultaneous analysis of 25OHD3 and 24,25(OH)2D3 both in human serum and cerebrospinal fluid by LC-MS/MS

Abstract

As a pleiotropic hormone, vitamin D (VD) is not only involved in bone health, but also associated with various disorders. Determination of circulating 25OHD₃ is routinely used to measure the status of VD, however previously reported methods exhibited various defects in accuracy, sensitivity, rapidness and simplicity in the analysis of VD and its metabolites. We present here a validated method for the simultaneous analysis of 25OHD₃ and 24,25(OH)₂D₃ both in low volumes of serum and cerebrospinal fluid (CSF) with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a deuterated internal standard. Serum and CSF samples were simply processed by protein precipitation and derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to enhance their responses and sensitivity. Separation was conducted using a gradient program on a Thermo Accucore C18 column. Mass spectrometry was operated using electrospray ionization (ESI) in the positive ion and multiple reaction monitoring (MRM) mode. The method exhibited excellent linearity with correlation coefficients above 0.99. The lower limits of quantification (LLOQ) for 25OHD₃ and 24,25(OH)₂D₃ were 0.10 and 0.25 ng mL⁻¹, respectively. Intra-day precision ranged from 1.4% to 9.7% and inter-day precision ranged from 1.9% to 10.0% for both 25OHD₃ and 24,25(OH)₂D₃. This method is precise, sensitive and simple, which is practical for the study of VD and its metabolites in human serum and CSF.