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Ann Clin Biochem. 2009 May;46(Pt 3):226-30. doi: 10.1258/acb.2009.008206.

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

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Department of Clinical Biochemistry, Macewen Building, Glasgow Royal Infirmary, Glasgow G4 0SF, UK.



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).


Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) 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 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode.


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.


Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) 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.

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