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 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 25OHD 3 (epi-25OHD 3 ) in human serum. Methods Tri- 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 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 × 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 395.3 > 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 2 , 25OHD 3 and epi-25OHD 3 were quantitated in 98 blood donors ( Conclusions This method is highly accurate, precise and specific.