Simultaneous profiling of serum vitamin D (VD) metabolites with similar structures is a big challenge. Thus, we developed and validated a SFC-MS/MS method, which is capable of eluting hydrophobic molecules, for quantification of VD2/VD3, 25-OH VD2/VD3, 3-epi-25-OH VD2/VD3, 1,25-(OH)2 VD2/VD3 and 24,25-(OH)2 VD2/VD3. VD metabolites were extracted from human serum using acetonitrile solvent. Column stationary phase, elution gradients, flow rate, column temperature, ion-source type and buffer system in post-column make-up solvent were optimized. Baseline separation of 10 VD metabolites can be achieved using PFP column within 10 min; and detection performed under positive electrospray ionization mode allowed quantification of VD metabolites in serum matrix with a limit of quantification (LOQ) varrying from 0.071 to 0.704 ng/mL. The accuracy was controlled with relative bias lower than 5.5% for QC and NIST samples. The developed method showed excellent intra-assay (0.52-7.93% RSD) and inter-assay (1.35-9.04% RSD) precision. The methodology shows enhanced efficiency and sensitivity as compared to LC-MS/MS method using the same column and mass spectrometer, along with significant correlation and low mean difference bias on measurements. For analysis of trace 1,25-(OH)2 VD2 and 1,25-(OH)2 VD3 in normal human serum or plasma, further improvement of LOQ (like derivatization) should be considered. In conclusion, the use of supercritical fluid not only enhanced safety with reduced solvent cost, but also improved retention and sensitivity as compared to LC-MS/MS method. The developed SFC-MS/MS method is appropriate for high throughput analysis of multiple VD metabolites in human serum with reduced solvent and economic cost.
Keywords: Metabolites; Serum; Supercritical fluid chromatography; Vitamin D.
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