|Sensitive and selective detection of Vitamin D3 and its metabolites 25 hydroxy vitamin D3 (25-OH-D3) and 1á 25-dihydroxyvitamin D3 (1,á25 (OH)2 D3) is critical for the diagnosis of vitamin D3 status (deficiency/insufficiency) and Calcium metabolic diseases. The normal levels of 25(OH) VD3 in human plasma are 32-72ng/mL however the active metabolite of VD3 ,1á 25(OH)2D3, is present in the plasma in low pg/mL ranges. The ionization efficiency of neutral molecules such as VD3 and its metabolites is very poor in Electrospray Mass Spectrometers (ESI/MS), making it impossible to quantify by this method in a reliable manner. Increasing the ionization efficiency of these compounds is crucial for a highly selective and reliable analysis.|
A novel derivatising reagent has been developed to derivatize VD3 and its metabolites in a simple and short one step reaction. The derivatized (charged) product yields much more intense fragments when subject to MS/MS collision energy when compared to the underivatised, thus increases significantly its electrospray sensitivity. It also creates a more hydrophilic nature being imparted on the molecule which results in shorter run times. A heavy version of the second reagent could be used as an internal standard for quantitation.
Analysis is performed on API 4000 LC/MS/MS system electrospary ion source, MRM (Multiple Reaction Monitoring) mode. The analytical chromatography is performed using a Phenomenex Luna C8 column 50x2.0mm, 5µm with a binary water/acetonitrile gradient of <10minutes. Using the specific MRM transitions for each derivatized metabolite, the detection limit (LOD) is increased >100 fold upon derivatization to levels of low pg/mL. The linearity and reproducibility of the method is currently addressed.
The derivatization technique presented herein is generic and could be used for a broader range of VD3 analogues and Vitamin D2