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

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 (100 μl) to monitor recovery. Liquid–liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8–100 nmol/L 25OHD_2, 12–150 nmol/L 25OHD₃, and 4–50 nmol/L epi-25OHD₃] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1 × 100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min 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 ≥ 20 nmol/l. Bias averaged < 5%. Detection limits were < 5 nmol/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.