Performances of a method for the quantification of 1alpha,25(OH)2-Vitamin D3 in serum by LC-MSMS without derivatization.
Mon 11:00 AM - Track 1: Small Molecule Analysis I
Bruno Casetta
Bruno Casetta, AB SCIEX Monza (Italy)
Adam Latawiec, AB SCIEX Toronto (Canada)
1alpha,25(OH)2-Vitamin D3 is the biologically active metabolite of vitamin D3 and is useful for the differential assessment of hyper- and hypo-calcemia, for distinguishing between vitamin D dependant and vitamin D resistant rickets, for monitoring vitamin D status of patients with chronic renal failure and for assessing compliance of 1alpha,25(OH)2-Vitamin D3 therapy.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been successfully used for the quantification of several steroid hormones and is nowadays considered as the gold standard method for measuring the serum 25-hydroxyvitamin D3 especially since accuracy of other methods for 25-hydroxyvitamin D3 are notoriously problematic
Quantification of 1alpha,25(OH)2-Vitamin D3 in serum is technically challenging because its very low concentration, at pg/mL level.
Even mass spectrometry faces the challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented.
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.

With an achieved limit of quantification (LOQ) of 15 pg/mL and with a % CV of 5-15 % at physiological concentration levels, the method has demonstrated to be very robust as long as the chromatographic set-up is correctly implemented.