A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry

Background: Measurement of 25-hydroxyvitamin D-3 (25OHD(3)) and D-2 (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE). Methods: Internal standard (hexadeuterated 25-hydroxyvitamin D3) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP (TM) SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. Results: The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D-3 and 17.5-70 nmol/L for D-2 (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias. Conclusions: Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD3 and D-2 in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention.