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

Background: An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D-2 (25OHD(2)), 25-hydroxyvitamin D-3 (25OHD(3)) and the C-3 epimer of 25OHD3 (epi-25OHD(3)) in human serum. Methods: Tri- and hexa-deuterated internal standards were added to serum (100 mu l) 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 25OHD(3), and 4-50 nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1 x 100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 3953>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2), m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3. Results: Recovery averaged >= 98%. Total imprecision was <= 10% when concentrations were >= 20 nmol/l. Bias averaged <5%. Detection limits were <5 nmol/l. Median (nmol/l) 250HD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (