Development of a method for the quantification of 1 alpha,25(OH)(2)-vitamin D-3 in serum by liquid chromatography tandem mass spectrometry without derivatization

摘要

Quantification of 1 alpha,25(OH)(2)-vitamin D-3 in serum is technically challenging because of its very tow concentration. Even mass spectrometry faces a challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented. Therefore, there is stilt a need for a robust, simplified, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 1 alpha,25(OH)(2)-vitamin D-3. Thanks to tandem mass-spectrometry associated to an on-tine 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.