Abstract

European Journal of Mass Spectrometry
Volume 16 Issue 1, Pages 81–89 (2010)
doi: 10.1255/ejms.1024

Development of a method for the quantification of 1α,25(OH)₂–vitamin D₃ in serum by liquid chromatography tandem mass spectrometry without derivatization

Bruno Casetta,^a Ivo Jans,^b Jaak Billen,^c Dirk Vanderschueren^b and Roger Bouillon^b
aApplied Biosystems, Monza, Italy. E-mail: bruno.casetta@eur.appliedbiosystems.com
^bClinic and Laboratory for Experimental Medicine and Endocrinology, Catholic University of Leuven, Belgium
^cDepartment of Laboratory Medicine, Catholic University of Leuven, Belgium

Quantification of 1α,25(OH)₂–vitamin D₃ in serum is technically challenging because of its very low concentration. Even mass spectrometry faces a challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented. Therefore, there is still a need for a robust, simplified, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 1α,25(OH)₂–vitamin D₃. Thanks to tandem mass-spectrometry associated to an on-line sample extraction and clean-up, sample preparation has been reduced to a simple protein precipitation step and derivatization has been avoided. Specimen volume has been kept to a minimum. The innovative aspects of the hereby-presented method are the use of stable lithium adducts and a highly sensitive tandem mass spectrometer. The achieved limit of quantification is at a level of 15 pg mL^–1, with a % CV of 5–15% at physiological concentration levels. The linearity is good and spiking experiments yielded a recovery of 87–102%. Comparison of selected samples with an established reference method, using an extensive purification procedure (high-performance liquid chromatography and radioimmunoassay), has shown a good correlation.

Keywords: 1α,25(OH)₂–vitamin D₃, on-line SPE, tandem mass spectrometry, electrospray ionization, perfusion chromatography, vitamin D

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