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

BackgroundMeasurement 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).MethodsInternal 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.ResultsThe 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)...