SUITABILITY OF 24,25(OH)2 VITAMIN D3 DETERMINATION WITH AN ADAPTED VERSION OF THE CHROMSYSTEMS® MASSCHROM® 25-OH-VITAMIN D3/D2– LC-MS/MS KIT

BACKGROUND: The enzym CYP24A1 catalyses the conversion of 25(OH)D3 in 24,25(OH)2D3. Recently, loss-of-function mutation of CYP24A1 has been identified in idiopathic infantile hypercalcemia (IIH). This genetic defect can be highlighted by high 1,25(OH)2D3 and undetectable 24,25(OH)2D3 levels. 24,25(OH)2D3 is also known to interfere with 25(OH)D3 determinations with immunoassays, leading to an overestimation of the 25(OH)D3 concentrations. We adapted
the MassChrom kit on the AB SCIEX TQ 5500 in order to systematically provide, next to 25(OH)D3, 25(OH)D2 and C3 epimer, the concentrations of 24,25(OH)2D3. The aim of this study was to evaluate the performance of 24,25(OH)2D3 determination with this modified method. We also wanted to establish the reference value of 24,25(OH)2D3.
METHODS: We modified the Chromsystems MassChrom method by adding the 24,25(OH)D3 correspondent transitions and performed a calibration by spiking known amounts of 24.25(OH)2D3. The LOQ was determined with 10 concentration levels of 24,25(OH)2D3. We selected 92 healthy children (40 girls; 2.4±1.51 years) presenting normal calcium levels (2.49±0.13mmol/l) to determine the 95th percentile (p95).
RESULTS: The 24,25(OH)2D3 LOQ was 4.7 ng/ml. 85.9% of our subjects were below this LOQ. The p 95 for the 24,25(OH)2D3, according to the CLSI C28-A3, was <6.2 ng/ml. The average serum concentrations (mean±SD) of 25(OH)D3 and 3-epi-25(OH)D3 were 24.48±10.22ng/ml and 2.07±1.86 ng/ml respectively. The 24,25(OH)2D3 levels (r2=0.64) correlated with the 25(OH)D3 levels.
CONCLUSIONS: Our adapted method from Chromsystems Vitamin D determination is available to quantify 24,25(OH)2D3. In this context, this method is able to determine high levels of 24,25(OH)2D3 that can possibly cross react with immunoassays. However, as the LOQ was not low enough, we couldn’t establish correct reference value for 24,25(OH)2D3. A derivatization step in the sample preparation would be interesting to improve the sensibility of the method.