LC/MS/MS Analysis of Vitamin D Metabolites on Ascentis® Express F5
Materials
analytical column
standard
25-Hydroxyvitamin D2 solution
50 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®25-Hydroxyvitamin D3 solution
100 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®1α,25-Dihydroxyvitamin D3 solution
5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®1α,25-Dihydroxyvitamin D2 solution
5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®CONDITIONS
column
Ascentis Express F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U)
mobile phase
[A] 5 mM ammonium formate water; [B] methanol; (25:75, A:B)
flow rate
0.4 mL/min
pressure
5178 psi (357 bar)
column temp.
40 °C
detector
ESI(+), 100-1000 m/z
injection
2 μL, each compound 300 ng/mL in 40:60 (water:methanol)
Description
Analysis Note
The metabolic role and health implications of the various vitamin D isoforms are of current clinical interest. Therefore, it is important to have an analytical method that will resolve all of the known isoforms with necessary sensitivity and specificity.This application shows an Ascentis Express F5 provided rapid resolution of the isobaric vitamin D metabolites. MS detection provides the necessary secondary resolution. The highest grade LC-MS solvents were used to supply low background interference and low particulate contaminants for robust, trouble-free operation. Cerilliant CRMs provided reliable identification and quantification.
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany