Background Measurement of 25-hydroxyvitamin D3 (25OHD3) and D2 (25OHD2) 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™ SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD3 and 25OHD2 was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. Results The lower limit of quantitation was 4.0 nmol/L for 25OHD3 and 7.5 nmol/L for 25OHD2. Within- and between-assay precision was below 10% over the concentration range of 22.5–120 nmol/L for D3 and 17.5–70 nmol/L for D2 ( 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 D2 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.
National Estimates of Serum Total 25-Hydroxyvitamin D and Metabolite Concentrations Measured by Liquid Chromatography–Tandem Mass Spectrometry in the US Population during 2007–2010
