Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum

Abstract

BackgroundAn ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) and the C-3 epimer of 25OHD3 (epi-25OHD3) in human serum. MethodsTri- and hexa-deuterated internal standards were added to serum (100μl) to monitor recovery. Liquid–liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8–100nmol/L 25OHD2, 12–150nmol/L 25OHD3, and 4–50nmol/L epi-25OHD3] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4ml/min, run time was 14min per sample; 25OHD3 and epi-25OHD3 were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD2, m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD3, m/z 383.3>365.3 (105.1 qualifier); d3-25OHD2, m/z 398.3>380.3; and d6-25OHD3, m/z 389.3>371.3. ResultsRecovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20nmol/l. Bias averaged <5%. Detection limits were <5nmol/l. Median (nmol/l) 25OHD2, 25OHD3 and epi-25OHD3 were quantitated in 98 blood donors (<LOD, 56.0, <LOD) and 35 pregnant women (<LOD, 87.6, 3.70). ConclusionsThis method is highly accurate, precise and specific.