|We developed a semi-automated method for the preparation of serum samples analyzed for 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3). Using a robotic system, 100 uL of serum is pipetted into a 96-well plate along with 75 uL water and 75 uL of a deuterated d3-25-OHD2 and d6-25-OHD3 internal standard mixture. The solution is mixed and transferred to a 96-well precipitation plate containing 300 uL acetonitrile and is further mixed. After 20 min, the precipitated protein is removed by centrifugal filtration into a 96-well receiving plate. Sixty uL of extract is injected onto a 2.1x30 Phenomenex C8(2) column and washed with 50% methanol:water. After 3.5 min, the mobile phase concentration is increased to 85% methanol and the analytes are eluted to a second HPLC column (2.1x50 Phenomenex C18(2) to achieve chromatographic resolution. Analytes are detected with a ThermoElectron Quantum Ultra mass spectrometer operating in positive ion atmospheric pressure chemical ionization mode. The MRM transitions (m/z) for 25-OHD2, 25-OHD3, and their respective internal standards are: 413>395, 401>383, 416>398, and 407>389. Each sample is analyzed in about 10 min, and a 96-well plate can be completed in less than 24 hours.|
Calibration curves were prepared from standards dissolved in 4% albumin-PBS and extracted in the same manner as serum samples. Calibrators ranged from 2.5-100 ng/mL. Quantitative results were obtained by plotting peak area ratios against concentration using weighted linear regression. Within-day analytical precision was determined using replicate analyses of serum samples (n=6) with mean 25-OHD3 concentrations of 12.4, 20.8, and 33.3 ng/mL with corresponding coefficients of variation (CV) of 1.5%, 2.1% and 0.9%. Between-day precision (n=20) for the same sera gave CVs of 7.8%, 5.2% and 4.9%, respectively. Similarly, mean within-day 25-OHD2 sample concentrations were 0.85, 2.73, and 8.95 ng/mL with corresponding CVs of 15.4%, 5.7% and 3.5%. Between-day CV were 32.2%, 10.4%, and 6.3%, respectively. Limits of detection, estimated using replicate analysis of serially diluted serum samples were 0.4 and 0.4 ng/mL for 25-OHD3 and 25-OHD2, respectively. Four levels of standard reference materials (SRM 972) showed good agreement with certificated values. NIST certified values for 25-OHD2: 0.6, 1.7, 26.4, and 2.4 ng/mL and for 25-OHD3: 25.3, 13.0, 19.6, and 70.7 ng/mL (sum of 25-OHD3 and C-3-epimer-25-OHD3). CDC between-day means (n=20) for 25-OHD2: 0.7 (117%), 2.2 (129%), 26.6 (101%), and 2.7 (112%) and for 25-OHD3: 26.1(103%), 13.1(101%), 20.0 (102%), and 74.9 (106%) ng/mL. Efforts to separate C-3-epimer-25OHD are underway.