Development of a Method for the Quantification of 1α,25(OH)₂–Vitamin D₃ in Serum by Liquid Chromatography Tandem Mass Spectrometry without Derivatization

Quantification of 1 α,25(OH) 2 –vitamin D 3 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 1 α,25(OH) 2 –vitamin D 3 . 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.