Simultaneous determination of 25(OH)D2, 25(OH)D3 and 1α,25(OH)2D3 in human serum by derivatization-liquid chromatography-tandem mass spectrometry

An LC-MS/MS method was developed and validated for simultaneously quantifying 25-hydroxyvitamin D2, D3, and 1α,25-dihydroxyvitamin D3 in human serum. Protein in 200 μL serum was precipitated with acetonitrile. After centrifugation, the metabolites were derivatized using (diacetoxyiodo)benzene (PIA) and 4-(4-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMAT) and then quantified by the LC-MS/MS system. The limits of detection (LODs) for the three substances were 10, 10, and 5 pg/mL, and the limits of quantification (LOQs) were 20, 20, and 10 pg/mL. The standard curves for these compounds showed linear regression coefficients (R2)>0.998 over specific concentration ranges. Recoveries were 94.36 %–102.34 % for 25(OH)D2, 92.43 %–103.41 % for 25(OH)D3, and 88.98 %–94.36 % for 1α,25(OH)2D3. The mean serum levels in 109 subjects (consisting of 61 healthy adult males and 59 healthy adult females) were 2.0 ± 1.5 ng/ml (25(OH)D2), 16.4 ± 6.1 ng/ml (25(OH)D3) and 36.6 ± 15.1 pg/ml (1α, 25(OH)2D3).Derivatization of vitamin D metabolites using PIA and DMAT is useful for rapidly determining the serum 25(OH) D2, 25(OH) D3 and 1α,25(OH)2D3 concentrations simultaneously in human serum.