Development of a method for the quantification of 1alpha,25(OH)2-vitamin D3 in serum by liquid chromatography tandem mass spectrometry without derivatization.
Quantification of 1alpha,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 1alpha,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.
Casetta
Bruno
B
Applied Biosystems, Monza, Italy. bruno.casetta@eur.appliedbiosystems.com
Jans
Ivo
I
Billen
Jaak
J
Vanderschueren
Dirk
D
Bouillon
Roger
R
D002762:Cholecalciferol / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002762:Cholecalciferol